Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1206 lipopolysaccharide containing D-allothreonine

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1206 lipopolysaccharide has been investigated. Methylation analysis, partial acid hydrolysis, FAB-MS/MS and 1H-NMR and 13C-NMR spectroscopy were the principal methods used. D-Allothreonine (D-aThr), amide-linked to the D-galacturonic acid, was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: [structure: see text].     

Structural studies of the O-specific chains of Hafnia alvei strains 744, PCM 1194 and PCM 1210 lipopolysaccharides

The structures of the O-specific chains of the Hafnia alvei strain 744 and PCM strains 1194 and 1210 lipopolysaccharides have been investigated. Methylation analysis, dephosphorylation, NMR spectroscopy, matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and fast-atom-bombardment mass spectrometry were the principal methods used. It was concluded that the polysaccharides of strains 744 and PCM 1194 are composed of the same pentasaccharide repeating unit having the following structure: [structure in text] and that the polysaccharide of strain PCM 1210 is composed of a pentasaccharide repeating unit having the following structure: [structure in text].     

NMR spectroscopic elucidation of the structure of a glycerol teichoic acid-like O-specific polysaccharide of the bacterium Hafnia alvei strain PCM 1199

The structure of the acidic O-specific polysaccharide of a Gram-negative bacterium, H. alvei strain PCM 1199, was studied by NMR spectroscopy including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), 1H, 13C heteronuclear single-quantum coherence (HSQC), 1H, 13C heteronuclear multiple-bond correlation (HMBC), and one-dimensional 1H, 31P heteronuclear multiple-quantum coherence (HMQC) experiments. It was found that the polysaccharide contains D-galactose, 2-acetamido-2-deoxy-D-glucose, 4-acetamido-4,6-dideoxy-D-glucose, glycerol, and phosphate in the ratios 1:2:1:1:1, as well as O-acetyl groups in non-stoichiometric amounts. The polysaccharide is similar in structure to teichoic acids of Gram-positive bacteria and has the following structure of the repeating unit: 3)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4NAc-(1- ->1)-Gro- 3-P-(O--> [formula: see text] beta-D-GlcpNAc [formula: see text] The O-specific polysaccharide of H. alvei PCM 1199 is structurally related to another teichoic acid-like O-specific polysaccharide of H. alvei PCM 1205 studied by us earlier.     

Structural and immunochemical studies on the O-specific polysaccharide of Proteus penneri strain 15

On the basis of sugar analysis and 1H- and 13C-NMR spectroscopy, it was shown that the O-specific polysaccharide of Proteus penneri strain 15 has a trisaccharide repeating unit, including an acetal-linked pyruvic acid residue, and is structurally identical to the capsular polysaccharide of Proteus vulgaris strain ATCC 49990. Serological studies supported this conclusion and demonstrated the presence in the homological antiserum of both anti-core and anti-O chain antibodies reacting with a lipopolysaccharide (LPS) epitope containing N-acetylglucosamine and galactose residues.     

Immunochemical studies of O-specific polysaccharide from Proteus penneri 14 lipopolysaccharide

OBJECTIVE: To assess the safety of Norplant contraceptive implant use by women with mild-moderate homozygous sickle cell disease (HbSS). METHOD: Prospective observation of women pre- and post-insertion of Norplant, with each woman serving as her own control. Participants: 25 women 18-40 years of age who attended a hospital sickle cell clinic; post-insertion data were available for 23 women. Outcome measures: Changes in hematologic parameters including PCV, MCV, reticulocytes, ISCs, HbF and bilirubin; changes in biochemical parameters including HDL cholesterol, aspartate transaminase, alkaline phosphate, serum creatinine and serum albumin. RESULT: With a mean follow-up of 12.4 months (range 1-29 months), there were no clinically or statistically significant group or individual changes in the hematologic or biochemical parameters after Norplant insertion. CONCLUSION: Norplant appears to be a safe and appropriate contraceptive for women with mild-moderate HbSS disease.     

Structural and immunologic studies of Proteus mirabilis 033 O-specific polysaccharide

A variety of adrenal imaging agents have been used in nuclear medicine, but no agent has been developed for magnetic resonance (MR) imaging. The authors have previously observed accumulation of aminated macromolecules in adrenal glands. They now report the synthesis of a model polymeric aminated contrast agent for enhanced MR imaging of the adrenal glands. The model agent consisted of a poly-L-lysine conjugate (molecular weight, 245 kd) that had 70% free epsilon amino groups and 30% diethylenetriaminepentaacetic acid (DTPA)-derivatized amino groups to bind indium-111 or gadolinium. One hour after intravenous administration of this compound, adrenal uptake was 10.1% +/- 0.7 of injected dose per gram of tissue. When all free epsilon amino groups of the polylysine were completely substituted with DTPA, adrenal uptake was 3.4 times lower, indicating the importance of free amino groups for adrenal uptake. MR imaging in rats showed that a dose of 0.08 mmol of gadolinium per kilogram of the agent was sufficient to enhance the signal intensity of adrenal glands. There hours after intravenous administration of the agent, signal intensity of the adrenal glands was 186% of precontrast values (liver, 165%; kidney, 91%). Fluorescence microscopy showed that the agent accumulated primarily in the cortical zona glomerulosa and in the adrenal medulla. These initial studies demonstrate the feasibility of designing contrast agents for MR imaging of the adrenal glands.     

3-Deoxy-octulosonic-acid-containing hexasaccharide fragment of unusual core type isolated from Hafnia alvei 2 lipopolysaccharide

The hexasaccharide containing 3-deoxy-octulosonic acid (Kdo) was isolated from the carbohydrate material obtained after the mild acid hydrolysis of lipopolysaccharide of Hafnia alvei strain 2. The hexasaccharide was purified by gel filtration on Bio-Gel P-4 and P-2 columns. On the basis of sugar and methylation analyses, one- and two-dimensional NMR spectroscopic methods, the hexasaccharide was identified as [formula: see text] The tetrasaccharide linked to position 7 of Kdo is also part of the sialic-acid-containing O-specific unit. Di-substitution of Kdo at positions 7 and 8 has not been previously reported.     

Structural studies of the Vibrio salmonicida lipopolysaccharide

The oligosaccharide part of the Vibrio salmonicida (strain NCMB 2262) lipopolysaccharide was isolated by mild acid hydrolysis followed by gel-permeation chromatography. The structure was established mainly by methylation analysis, mass spectrometry, and NMR spectroscopy. It is concluded that the oligosaccharide has the following structure, in which L-alpha-D-Hep p is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hepp is D-glycero-alpha-D-manno-heptopyranose, alpha-D-Fuc p4N is 4-amino-4,6-dideoxy-alpha-D-galactopyranose, alpha-NonA is 5-acetamidino-7-acetamido-3,5,7, 9-tetradeoxy-L-glycero-alpha-D-galacto-nonulosonic acid, BA is (R)-3-hydroxybutanoyl, and PEA is phosphoethanolamine. The substitution pattern of the branching heptosyl residue was deduced from 1H NMR chemical shifts and conformations of the branching region, obtained by molecular modelling. The absolute configuration for NonA was determined by NMR spectroscopy from NOE correlations to the neighbouring sugar and 13C NMR chemical shift data. It could also be shown that assignments of nonulosonic acids with the D-glycero-L-galacto configuration, reported by previous investigators, are erroneous and should be changed to L-glycero-D-galacto. The oligosaccharide is assumed to be linked to the 5-position of a Kdo residue, phosphorylated in the 4-position as observed for other lipopolysaccharides from Vibrionaceae. [formula: see text]     

Heterogeneity in phenotypic and genotypic characteristics among strains of Hafnia alvei

Hafnia alvei is an emerging human pathogen associated with sporadic cases and outbreaks of diarrhea. Bangladeshi isolates of H. alvei possess the Escherichia coli attaching and effacing (eaeA) gene and demonstrate an attaching and effacing phenotype. In the present study we examined 11 Canadian H. alvei isolates and strain 19,982 from Bangladesh to determine if the formation of attaching and effacing lesions is a property shared among multiple isolates. Attaching and effacing lesions were detected by induction of tyrosine kinase protein phosphorylation and cytoskeletal rearrangements in infected tissue culture epithelial cells with immunofluorescence microscopy and by the examination of infected cells with transmission electron microscopy. The presence of the eaeA gene was examined by PCR and colony blot hybridization. Profiles of outer membrane protein extracts, chromosomal macrorestriction fragments, and plasmids were also examined. Accumulation of host phosphotyrosine proteins and rearrangement of the cytoskeletal protein alpha-actinin were both observed in HEp-2 cells infected with H. alvei 19,982. In contrast, none of the other 11 clinical H. alvei isolates demonstrated either of these responses, nor did they form attaching and effacing lesions under electron microscopy. Consistent with the absence of the attaching and effacing phenotype, these clinical isolates did not possess the eaeA gene. The outer membrane protein profiles of all the Canadian isolates were identical but differed from that of H. alvei 19,982. Pulsed-field gel electrophoresis and plasmid profile analyses of the clinical H. alvei isolates differed substantially from those of the Bangladeshi strain. These results indicate that there is heterogeneity among H. alvei strains with respect to signal transduction, attaching and effacing adhesion, outer membrane constituents, and genotype. Epidemiological studies on enteropathogenic H. alvei thus need to go beyond simple species designations and require specific identification of the virulent clones.     

Structural characterization of the O-antigenic polysaccharide of Escherichia coli serotype 017 lipopolysaccharide

Transverse nuclear magnetic relaxation and self-diffusion of water were measured in hydrated collagen II. Self-diffusion measurements were conducted by pulsed field gradient NMR (PFG NMR) and weighting of the different species in the signal by variable T2 relaxation in the experiment. Two fractions of water protons were detected, one with a short T2 value but high diffusivity and one with a long T2 value and low, completely restricted diffusion. The distance of the diffusion barriers was determined to be 2.3 microns. Possible reasons for the restriction in the movement of the water molecules in comparison with structural models of collagen II are discussed.     

The O-specific polysaccharide chain of Campylobacter fetus serotype A lipopolysaccharide is a partially O-acetylated 1,3-linked alpha-D-mannan

A polysaccharide fraction liberated from Campylobacter fetus subsp. fetus serotype A lipopolysaccharide by mild acid hydrolysis followed by gel-permeation chromatography contained a partially O-acetylated D-mannan chain, as an O-specific polysaccharide, with a core oligosaccharide attached. The structure of the polysaccharide was studied by O-deacetylation, methylation, and 1H- and 13C-NMR spectroscopy, including computer-assisted analysis of the 13C-NMR spectrum. A structure of -->3)-alpha-D-Manp2Ac-(1--> was established as the structure of the O-specific polysaccharide, the degree of O-acetylation of the mannose residues at position 2 being estimated as 80-90%. As judged by the ratio of mannose to core constituents, the D-mannan chain consists on average of 10-12 monosaccharide units.     

Structural studies of the putative O-specific polysaccharide of Acinetobacter baumannii O2 containing 3,6-dideoxy-3-N-(D-3-hydroxybutyryl)amino-D-galactose

A polysaccharide containing D-galactose, 2-deoxy-2-N-acetylamino-D-galactose and 3,6-dideoxy-3-N-(D-3-hydroxybutyryl)amino-D-galactose, probably corresponding to the lipopolysaccharide side chain, was obtained from an aqueous phenol extract of isolated cell walls from Acinetobacter baumannii strain O2. By means of NMR studies and chemical degradations, the repeating unit of the polymer was identified as a branched hexasaccharide of the structure shown, where Fuc3N represents 3-amino-3,6-dideoxygalactose and R represents D-3-hydroxybutyryl. Serological tests indicated that the polymer corresponded to the O2 antigen.     

Clinical significance of extraintestinal Hafnia alvei isolates from 61 patients and review of the literature

Hafnia alvei is a gram-negative bacterium that is rarely isolated from human specimens and is rarely considered to be pathogenic. It has been associated with gastroenteritis, meningitis, bacteremia, pneumonia, nosocomial wound infections, endophthalmitis, and a buttock abscess. We studied 80 H. alvei isolates recovered from 61 patients within a period of 30 months. H. alvei was cultured from sites that included the respiratory tract (n = 38), the gastrointestinal tract (n = 16), and the urogenital tract (n = 12); the organism was found in blood cultures (n = 8), on central venous catheters (n = 3), and on the skin (n = 3). Only 25% of H. alvei isolates were recovered in pure cultures. Fifty-seven (93.4%) of the patients had an underlying illness. H. alvei proved to be the etiologic agent in two episodes of septicemia and in one episode of peritonitis and was probably responsible for septicemia in two other patients and pneumonia in one. All six of these patients recovered after receiving antibiotic treatment and/or standard surgical treatment, when needed. Three of these infections were nosocomial, and three were community acquired. Of the strains of H. alvei tested in our study, 100% were susceptible to netilmicin, ciprofloxacin, and imipenem; 92% were susceptible to piperacillin; 90% were susceptible to co-trimoxazole; and 88% were susceptible to ceftriaxone and ceftazidime. In this study, we found H. alvei to be a rare significant etiologic agent of nosocomial and community-acquired infections.     

Structural studies of the O-antigenic polysaccharide of Fusobacterium necrophorum

The O-specific polysaccharide component of the lipopolysaccharide produced by Fusobacterium necrophorum is of the teichoic acid type, with repeating units connected by phosphoric diester linkages. Dephosphorylation of the polysaccharide by treatment with aqueous hydrogen fluoride yielded a carbohydrate composed of a trisaccharide linked to an acidic component. This product, and the polysaccharide, were investigated by chemical methods and 1H-, 13C-, 31P- and 15N-NMR spectroscopy and the former also by fast-atom-bombardment mass spectrometry. It is proposed that the polysaccharide is composed of repeating units having the following structure, in which Fuc represents fucose (6-deoxy-galactose), Am represents an acetamidino group and Sug 2,4-diamino-2,4,6-trideoxy-D-glucose ('bacillosamine') acetylated at the 2-position and acylated with a (S)-3-hydroxybutanoic acid at the 4-position. The acid was identified as a 2-amino-2-deoxy-2-C-methyl-pentonic acid (2-amino-2-methyl-3,4,5-trihydroxypentanoic acid). The configuration of this acid remains to be determined. [formula: see text]     

Structure of the O-specific polysaccharide of an Aeromonas trota strain cross-reactive with Vibrio cholerae O139 Bengal

The O-specific polysaccharide of an Aeromonas trota strain was isolated by hydrolysis of the lipopolysaccharide at pH 4.5 followed by gel-permeation chromatography and found to consist of hexasaccharide repeating units containing D-galactose, L-rhamnose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose in the ratios 1:1:2:1:1. Partial hydrolysis of the polysaccharide with 48% hydrofluoric acid resulted in selective removal of colitose to give a modified polysaccharide containing the other four sugar constituents. On the basis of methylation analysis and NMR spectroscopic studies of the initial and modified, colitose-free polysaccharide, it was concluded that the repeating unit of the O-specific polysaccharide has the following structure [sequence: see text] The known cross-reactivity between the strain studied and Vibrio cholerae O139 Bengal is substantiated by the presence of a common colitose-containing epitope shared by the O-specific polysaccharide of A. trota and the capsular polysaccharide of V. cholerae, which is thought to carry determinants of O-specificity.     

Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247

The O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247, was studied by acid hydrolysis, GLC-MS and 1H and 13C NMR spectroscopy, including 1D and 2D NOE, 2D hybrid TOCSY and ROESY (TORO), and 2D H-detected heteronuclear multiple-bond correlation (HMBC) experiments. The polysaccharide was found to contain L-rhamnose, 3.6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido- 2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb) and 2-acetamido-2- deoxy-D-galacturonic acid (D-GalNAcA). Partial acid hydrolysis of the polysaccharide resulted in a non-reducing GalNAcA-->QuiNAc4NHb disaccharide with the 3-hydroxybutyryl group glycosylated intramolecularly by the QuiN4N residue. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:-->4) -alpha-D-GalpNAcA-(1-->3)- alpha-D-QuipNAc4NHb-(1-->2)-beta-D-Quip3NHb-(1-->2)-alpha-L- Rhap(1-->.     

Structural studies of the O-antigen isolated from the phenol-soluble lipopolysaccharide of Acinetobacter baumannii (DNA group 2) strain 9

In Weaver mutants (B6CBA wv/wv) cerebellar granule cells degenerate almost completely postnatally. A partial loss of Purkinje cells (PC) and a degeneration of dopaminergic cells in the substantia nigra have also been found. Weaver mice suffer from striking motor symptoms, including difficulty in walking without toppling over. In an attempt to influence the poor motor performance, the cerebellum in young animals was removed, thus eliminating the faulty output of surviving PCs, demonstrated electrophysiologically. Unoperated Weaver, lesioned wildtypes and one sham mouse were used as controls. Before and after operation, a battery of behavioural tests was performed. In Weaver mice, tumbling to the side (t) and the relation of t to the motor activity (k) while traversing an open-field matrix, (t/k), improved considerably, as did manoeuvring on a slanted wire mesh, but keeping balance on a wooden bench did not to the same degree. Locomotor activity alone improved in some animals. In wildtypes no significant changes occurred after operation, with the exception of a strong reduction in locomotor activity. The experiments demonstrate that the motor performance in Weaver mutant mice benefits from removal of their cerebellum.     

The O-specific polysaccharide chain of Campylobacter fetus serotype B lipopolysaccharide is a D-rhamnan terminated with 3-O-methyl-D-rhamnose (D-acofriose)

An O-specific polysaccharide was liberated from Campylobacter fetus subsp. fetus serotype B lipopolysaccharide by mild acid hydrolysis followed by gel chromatography. This polysaccharide was found to contain D-rhamnose and 3-O-methyl-D-rhamnose (D-Rha3Me, D-acofriose) in a ratio of approximately 24:1, as well as lipopolysaccharide core constituents. The structure of the polysaccharide was studied by 1H-NMR and 13C-NMR spectroscopy, which included two-dimensional COSY, rotating-frame NOE spectroscopy (ROESY), and computer-assisted analysis of the 13C-NMR spectrum. Methylation analysis using [2H3]methyl iodide and Smith degradation followed by GLC/MS of the derived acetylated oligosaccharide-alditols was used to determine the location of D-acofriose. The O-specific polysaccharide is linear, consists on average of 12 disaccharide repeating units, and is terminated by a residue of D-acofriose. The following structure of the D-rhamnan chain was established: [sequence: see text]     

Structural elucidation of the capsular polysaccharide of Bacteroides fragilis strain 23745M1

The capsule of Bacteroides fragilis (ATCC23745) consists of two distinct polysaccharides, the separation of which could not be accomplished. The mouse-passaged strain (23745M1), however, yielded a preponderant polysaccharide which was isolated and purified. Using mainly high resolution NMR spectroscopy, the structure of the polysaccharide was elucidated and it is composed of the following repeating unit: [formula see text]     

Structural studies of the O-antigenic polysaccharide from Escherichia coli O167

The structure of the O-antigenic polysaccharide from Escherichia coli O167:H5 has been investigated. Sugar and methylation analyses, fast-atom-bombardment mass spectrometry and 1H- and 13C-NMR spectroscopy were the main methods used. The structure of the repeating unit of the polysaccharide was found to be: [formula in text]. Oligosaccharide derivatives of the polysaccharide were obtained by HF solvolysis and by a Smith degradation. Furthermore, base treatment of the polysaccharide led to a degraded polymeric material. For the methylated polysaccharide the amide linkage between alanine and the galacturonic acid residue was reductively cleaved with LiBD4 in ethanol, to give, among other things, a 3-O-methyl galactose derivative.