Structure of the lipopolysaccharide antigenic O-chain produced by Actinobacillus pleuropneumoniae serotype 4 (ATCC 33 378)

The structure of the antigenic O-polysaccharide part of the S-type lipopolysaccharide produced by Actinobacillus pleuropneumoniae serotype 4 has been determined by periodate oxidation, methylation, partial hydrolysis, and 1H- and 13C-n.m.r. spectroscopy. The O-polysaccharide structure has a branched-tetrasaccharide repeating unit, (----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----3)]-beta-D-GalpNAc- (1----4)-alpha-L-Rhap-(1-)n. The structure resembles that of the lipopolysaccharide O-chain of A. pleuropneumoniae serotype 7, and their common epitopes may account for the apparent serological cross-reactivity observed between the two serotypes when incompletely adsorbed, anticapsular-typing sera are used.     

Characterisation of the Salmonella O:8 antigen in the O-chain of the lipopolysaccharide produced by Salmonella virginia O:8.

The antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella virginia (O:8), analyzed by methylation, partial acid hydrolysis, and one- and two-dimensional nuclear magnetic resonance methods, was found to be a polymer of a repeating pentasaccharide unit composed of D-mannose, D-galactose, L-rhamnose, D-abequose, and O-acetyl (2:1:1:1:1.3) and having the following structure: [formula; see text] The disaccharide structure alpha-D-Abep-(1----3)-L-Rhap was identified as the Salmonella O:8 antigenic factor epitope, since the removal of alpha-D-Abep residues from the O-polysaccharide left a residual tetrasaccharide repeating unit backbone that did not show reaction with Salmonella type O:8 factor antiserum.     

Structure of the antigenic O-polysaccharide of the lipopolysaccharide produced by Salmonella eimsbuttel

The smooth lipopolysaccharide produced by Salmonella eimsbuttel, which had the O:6, O:7, and O:14 antigenic factors defined in the Kauffmann-White classification, was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, composition analysis, methylation, periodate oxidation, deamination, and 1H and 13C nuclear magnetic resonance studies to contain a high molecular weight O-chain polysaccharide composed of D-mannose (four parts), D-glucose (one part), and 2-acetamido-2-deoxy-D-glucose (one part). It was a branched polymer of a repeating hexasaccharide unit having the structure (formula; see text).     

Structure of the lipopolysaccharide O-chain of Yersinia enterocolitica serotype O:5,27

The cellular lipopolysaccharide produced by Yersinia enterocolitica serotype O:5,27 was of the S-type and composed of an antigenic O-chain polysaccharide linked through a core oligosaccharide region, which in turn was linked through 3-deoxy-D-manno-octulonosyl units to a lipid A moiety. The O-chain polysaccharide was composed of equal molar amounts of L-rhamnose and D-xylulose. By partial hydrolysis, periodate oxidation, methylation, specific optical rotation, and 13C and 1H nuclear magnetic resonance studies, the structure of the O-chain was established as being a linear backbone of alternating 1,3-linked alpha-L-rhamnopyranosyl and beta-L-rhamnopyranosyl units, to which 2,2-linked beta-D-threo-pent-2-ulofuranoside (D-xylulofuranoside) units were present on every L-rhamnopyranosyl residue, as shown below. (Formula: see text)     

Structural characterization of the antigenic O-chain of the lipopolysaccharide of Escherichia coli serotype O65

The antigenic O-polysaccharide moiety of the lipopolysaccharide produced by Escherichia coli serotype O65 was investigated by composition, methylation, base hydrolysis, periodate oxidation, mass spectrometric methods, and by 1D and 2D NMR spectroscopy. The O-polysaccharide had [alpha]D + 108 degrees (water) and is a high-molecular-weight unbranched linear polymer of repeating pentasaccharide units composed of 1:1:1:1:1 D-galacturonic acid (D-GalA), D-galacturonamide (D-GalANH2), 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galactose (D-GalNAc), and 3-acetamido-3,6-dideoxy-D-glucose (D-Qui3NAc), and has the following structure: [formula: see text]     

Structural characterization of the antigenic capsular polysaccharide and lipopolysaccharide O-chain produced by Actinobacillus pleuropneumoniae serotype 15.

The specific capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 15 was determined to be a high-molecular-mass polymer having [alpha]D + 69 degrees (water) and composed of a linear backbone of phosphate diester linked disaccharide units of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc) and 2-acetamido-2-deoxy-D-galactose (D-GalNAc) residues (1:1). Thirty percent of the D-GalNAc residues were substituted at O-4 by beta-D-galactopyranose (beta-D-Galp) residues. Through the application of chemical and NMR methods, the capsule, which defines the serotype specificity of the bacterium, was found to have the structure [structure: see text]. The O-polysaccharide (O-PS) component of the A. pleuro pneumoniae serotype 15 lipopolysaccharide (LPS) was characterized as a linear unbranched polymer of repeating pentasaccharide units composed of D-glucose (2 parts) and D-galactose (3 parts), shown to have the structure [structure: see text]. The O-PS was chemically identical with the O-antigen previously identified in the LPSs produced by A. pleuro pneumoniae serotypes 3 and 8.     

Structure of the O-chain of the lipopolysaccharide of Haemophilus pleuropneumoniae serotype 1

By phenol-water extraction an aqueous-phase soluble cellular lipopolysaccharide was isolated from Haemophilus pleuro-pneumoniae serotype 1. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hydrolysis, methylation, and both one- and two-dimensional 1H and 13C nuclear magnetic resonance studies to be an S-type lipopolysaccharide, which could be cleaved to yield a lipid A and an O-chain polysaccharide identified as a high molecular weight branched polymer of a tetrasaccharide repeating unit having the structure: (Formula: see text).     

The structures of the two lipopolysaccharide O-chains produced by Salmonella boecker

Salmonella boecker, which belongs to group 0:6, 14(H) and shows the antigenic factors 6, 14, [1], and [25], defined by the Kauffmann-White system, produces two lipopolysaccharides differing from each other in the structures of their 0-poly-saccharide moieties. By glycose composition, partial hydrolysis, nitrous acid deamination, methylation, optical rotation, and 1H and 13C nuclear magnetic resonance studies, the O-polysaccharides were demonstrated to be high-molecular-weight polymers (I and II) composed of either structurally related repeating tetrasaccharide or repeating pentasaccharide units having the structures and (table; see text).     

Structural characterization of the O-polysaccharide of the lipopolysaccharide produced by Salmonella milwaukee O:43 (group U) which possesses human blood group B activity.

The O-polysaccharide of the lipopolysaccharide produced by Salmonella milwaukee O:43 (group U) was shown by composition analysis, methylation, periodate oxidation, and 1H and 13C nuclear magnetic resonance spectroscopic analytical methods to be a polymer of branched pentasaccharide repeating units having the structure: [formula: see text] The blood-group activity of the O-polysaccharide was established by its serological reactivity with a specific monoclonal antibody to human blood group B, using passive hemagglutination and ELISA assays, indicating the common antigenic epitope to be a nonreducing terminal trisaccharide unit composed of L-Fucp and D-Galp (1:2) residues.     

Structure of the O-chain of the lipopolysaccharide of Pasteurella haemolytica serotype T3

Cell-wall lipopolysaccharide isolated from Pasteurella haemolytica serotype T3 using the phenol-water extraction procedure was shown to be an S type lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hydrolysis with mild acid afforded a lipid-free, antigenic O-chain polysaccharide. On the basis of one- and two-dimensional 1H and 13C nuclear magnetic resonance studies, in conjunction with microanalytical chemical methods, the O-polysaccharide was determined to be a linear polymer of a disaccharide repeating unit having the structure. [----3)-beta-D-G1cpNAc-(1----4)-alpha-L-Rhap-(1----]n     

Genetic determination of the O antigens of Salmonella groups B (4,5,12) and C1(6,7)

M?kel?, P. Helena (University of Helsinki, Helsinki, Finland). Genetic determination of the O antigens of Salmonella groups B (4,5,12) and C(1) (6,7). J. Bacteriol. 91:1115-1125. 1966.-The genetic determination of the O antigens of Salmonella was studied by Hfr or F' crosses between strains of groups B (antigens 4,5,12) and C(1) (antigens 6,7). The main genetic determinants of the specificities 4 of group B and 7 of group C(1) behaved as alleles of one locus, called O or O-4/7. This is probably identical with O-4/9, responsible for the serological difference between groups B and D, and with the "rough" locus rouB. At least parts of the antigens 12 of group B and 6(2) of group C are determined at the same locus. The gene O-5 is closely linked to O-4/7, both mapping in the approximately 2 minutes distance between his and metG in the order his - O-4/7 - O-5 - metG. In crosses of group B donors with group C(1) recipients, a serologically new type, called semirough (SR), appeared in most recombinants that had inherited the O-4 allele of the group B donor. These SR forms are serologically intermediate between smooth and rough forms, showing poor stability in saline but possessing the specificities 4 and 12 and (some of them) 5. On the basis of previous biochemical studies, the hypothesis has been put forward that the side chains of their lipopolysaccharide are much shorter than normal group B side chains, probably containing only one repeating unit per side chain. A gene SR-4 responsible for the elongation of group B side chains beyond the first repeating unit was mapped between gal and try, group B and D bacteria being Sr-4(+), and group C being SR-4(-).     

Efficient synthesis of a heptasaccharide,the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642

alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[beta-D-Xylp-(1-->2)-][beta-D-Xylp-(1-->4)-]alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642 was synthesized as its methyl glycoside via 3-O-selective glycosylation of methyl alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (9) with 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (8), followed by dixylosylation with 2,3,4-tri-O-benzoyl-alpha,beta-D-xylopyranosyl trichloroacetimidate (12) and subsequent deacylation.     

Clinical and veterinary isolates of Salmonella enterica serovar enteritidis defective in lipopolysaccharide O-chain polymerization

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and the O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, we analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.     

The structure of the O-polysaccharide isolated from the lipopolysaccharide of Salmonella Dakar (serogroup O:28)

The structure of the O-antigenic part of the lipopolysaccharide (LPS) of Salmonella Dakar was analysed using chemical methods, NMR spectroscopy and mass spectrometry. The following structure for the repeating unit of the O-polysaccharide was determined: [see text] where Quip3NAc is 3-acetamido-3,6-dideoxyglucose. This is the first published structure of the O-polysaccharides from 101 serotypes of Salmonella bacteria belonging to serogroup O:28 (formerly M) in the Kauffmann-White scheme.     

Characterization of the common antigenic lipopolysaccharide O-chains produced by Bordetella bronchiseptica and Bordetella parapertussis

Porins of Salmonella minnesota, R595, were purified by anion exchange chromatography and subsequently isolated in their monomeric form by chromatofocusing. Two forms of porin could be isolated, both with an apparent molecular mass of 37,000, but of differing isoelectric points (pI 4.6 versus pI of 4.9). Porins with pI 4.9 did not contain any detectable LPS, but porins with pI 4.6 were found to contain trace amounts of LPS (1.3 x 10(-4) micrograms LPS/1 microgram porin) as measured using a highly sensitive limulus assay. Unlike the LPS-associated porins the monomeric porins were biologically inert with regard to pore formation, but they were still able to bind C1q, a subcomponent of the first component of complement.     

Structural characterization of the antigenic O-polysaccharide in the lipopolysaccharide produced by Actinobacillus pleuropneumoniae serotype 14

The antigenic O-polysaccharide of the lipopolysaccharide produced by Actinobacillus pleuropneumoniae serotype 14 was shown by chemical analysis and 1D and 2D nuclear magnetic resonance methods to be a high-molecular-mass polymer of a repeating disaccharide unit composed of a chain of (1-->5)-linked beta-D-galactofuranose (beta-D-Galf) residues substituted at their O-2 positions by alpha-D-galactopyranose residues (D-Galp) (1:1): [formula: see text].     

Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7

The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol-water extraction procedure was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure: (formula; see text) The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4-amino-4, 6-dideoxy-alpha-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides.     

Structure of the sialic acid-containing O-specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide.

Lipopolysaccharide was extracted from cells of Salmonella enterica serovar Toucra O48 and, after mild acid hydrolysis (1% AcOH, 1 h, 100 degrees C or 0.1 M NaOH-AcOH, pH 4.5, 5 h, 100 degrees C), the O-specific polysaccharide was isolated and characterized. The core and an oligosaccharide containing a fragment of the repeating unit linked to the core region were also obtained, depending on hydrolysis conditions. On the basis of sugar and methylation analyses and NMR spectroscopy of the hydrolysis products, the biological repeating unit of the O-specific polysaccharide was shown to be the following trisaccharide: -->4)-alpha-Neup5Ac(2-->3)-L-alpha-FucpNAc(1-->3)-D-beta-Glc pNAc(1--> The polysaccharide O-chain was substituted with a single molar equivalent of O-acetyl group, distributed between the Neu5Ac O-9 and O-7 positions, in an approximate ratio of 7 : 3.