Interleukin-1 induction by lipopolysaccharides: structural requirements of the 3-deoxy-D-manno-2-octulosonic acid (KDO).

We previously showed the importance of the 3-deoxy-D-manno-2-octulosonic acid (KDO) residue in endotoxins (lipopolysaccharides, LPS) for the induction of the synthesis and release of interleukin-1 (IL-1) by human monocytes. We further investigated the effect of some structural variations within the KDO molecule on IL-1 production induced by LPS. Deamination of Bordetella pertussis LPS, followed by mild anhydrous acidic methanolysis released a hexasaccharide (fragment B'), which had a terminal methyl ketoside KDO residue with a methyl-esterified carboxyl group. This fragment was unable to induce IL-1 production by human monocytes. Fragment B' could be converted into an active hexasaccharide by de-esterification (fragment B-OMe), but not by reduction of the methyl ester group. The KDO residues in the LPS of some bacterial species have been shown to be phosphorylated and we observed that these LPS were weak IL-1 inducers. Phosphorylated KDO present in Vibrio cholerae and B. pertussis LPS respond poorly in the thiobarbiturate assay (specific for KDO). However, if these LPS were dephosphorylated with aqueous hydrofluoric acid (HF) their KDO response in this assay was increased 5.4- to 2.6-fold, respectively. In parallel, the HF-treated LPS were more potent IL-1 inducers than untreated endotoxins. These data confirm that the KDO residue(s) present in all endotoxins play(s) a major role in the signal(s) leading to IL-1 production by human monocytes, and show that IL-1 induction by LPS (1) requires a free carboxyl group in the KDO and (2) is correlated with the degree of substitution of the KDO.

Structural variability and originality of the Bordetella endotoxins.

Structural studies of Bordetella endotoxins (LPSs) have revealed remarkable differences: (i) between their LPSs and those of other bacterial pathogens; (ii) among the LPSs of the seven identified Bordetella species; and (iii) among the LPSs of some Bordetella strains. The lipid As have the "classical" bisphosphorylated diglucosamine backbone but tend to have fewer and species-specific fatty acid components compared to those of other genera. Nevertheless, three strains of B. bronchiseptica have at least three different fatty acid distributions; however, the recently identified B. hinzii and B. trematum LPSs had identical lipid A structures. The B. pertussis core is a dodecasaccharide multi-branched structure bearing amino and carboxylic groups. Another unusual feature is the presence of free amino sugars in the central core region and a complex distal trisaccharide unit containing five amino groups of which four are acetylated and one is methylated. The B. pertussis LPS does not have O-chains and that of B. trematum had only a single O-unit, unlike the LPSs of all the other species of the smooth-type. The O-chain-free cores of non-B. pertussis LPSs were always built on the B. pertussis core model but most were species-specifically incomplete. The LPS structures of three B. bronchiseptica strains were found to be different from each other. The O-chains of B. bronchiseptica and B. parapertussis were almost identical and had some features in common with B. hinzii O-chain. Serological analyses are consistent with the determined LPS structures.

Structure of the Bordetella pertussis 1414 endotoxin.

The endotoxin (lipopolysaccharide) of Bordetella pertussis, the agent of whooping cough, consists of a lipid A linked to a highly branched dodecasaccharide containing several acid and amino sugars. The elucidation of the polysaccharide structure was accomplished by first analyzing the structures of fragments obtained by hydrolysis and nitrous deamination and then piecing the fragments together. The fine structure of the antigenic distal pentasaccharide, presented here, was determined by chemical analyses as well as by high-resolution nuclear magnetic resonance and mass spectrometry. The complete structure was reconstituted and confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The following structure was derived from the combined experimental data:The detailed structure combined with previously reported serological data now allows the synthesis of its epitopes for potential vaccines.

Helicobacter mustelae lipid A structure differs from that of Helicobacter pylori.

The lipid A structure of the Gram-negative bacterium Helicobacter mustelae, a ferret gastric pathogen responsible for the onset of gastric diseases in its host, was investigated. Two variant lipid A structures were found in the same strain. One structure contained a bisphosphorylated beta-(1-->6)-linked D-glucosamine backbone disaccharide with hydroxytetradecanoic acid in amide linkages. Unlike the structure described for the lipid A of the related human Helicobacter pylori gastric pathogen, which contains a C1 phosphate moiety, this lipid A presented phosphate groups at both the C1 and C4' positions, and contained no octadecanoyl fatty acid, which is present in H. pylori. The second lipid A structure had a different fatty acid composition in that 3-OH C(16) replaced most of the amide-linked 3-OH C(14).

Novel variation of lipid A structures in strains of different Yersinia species.

The Yersinia genus includes human and animal pathogens (plague, enterocolitis). The fine structures of the endotoxin lipids A of seven strains of Yersinia enterocolitica, Yersinia ruckeri and Yersinia pestis were determined and compared using mass spectrometry. These lipids differed in secondary acylation at C-2': this was dodecanoic acid (C(12)) for two strains of Y. enterocolitica and Y. ruckeri, tetradecanoic acid (C(14)) in two other Y. enterocolitica and hexadecenoic acid (C(16:1)) in Y. pestis. The enterocolitica lipids having a mass identical to that of Escherichia coli were found to be structurally different. The results supported the idea of a relation between membrane fluidity and environmental adaptability in Yersinia.

Chemical and serological characterization of the Bordetella hinzii lipopolysaccharides.

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The polysaccharides isolated from its endotoxin (lipopolysaccharide, LPS) were investigated using chemical analyses, NMR, gas-liquid chromatography/mass spectrometry and mass spectrometry by plasma desorption, matrix-assisted laser desorption/ionization and electrospray. The following structure for the O-chain-free LPS was deduced from the experimental results: carbohydrate structure [see text] Mass spectrometry and serology revealed that the O-chains were different from the homopolymer common to Bordetella bronchiseptica and Bordetella parapertussis strains and were composed of a trisaccharide repeating unit. Masses up to 8 kDa were obtained for native LPS molecular species.

Dopamine B hydroxylase deficiency responsible for severe dysautonomic orthostatic hypotension in an elderly patient.

We report the case of an elderly woman with severe dysautonomic orthostatic hypotension in whom a deficit in dopamine B hydroxylase has been established. In the literature, such a deficit has been described in six young adults with long standing symptoms of postural hypotension. This enzyme catalyses the conversion of dopamine to noradrenaline. In our elderly patient, noradrenaline and adrenaline were undetectable in the plasma, but plasma dopamine was detectable. Treatment with the synthetic amino acid, DL-threo-dihydroxyphenylserine, which is converted to noradrenaline by dopa-decarboxylase, resulted in a significant increase in blood pressure. The mechanism of this acquired deficit is not elucidated.

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

Representative strains of Bordetella bronchiseptica and B. parapertussis were found to produce smooth lipopolysaccharides (LPS) having identical antigenic O-polysaccharide components composed of linear unbranched polymers of 1,4-linked 2,3-diacetamido-2,3-dideoxy-alpha-L-galacto-pyranosyluronic acid residues. These LPSs differed from the LPS of B. pertussis which produces only rough-type LPS, devoid of O-polysaccharide. While B. bronchiseptica and B. parapertussis had chemically and immunologically identical O-polysaccharide structures, their core oligosaccharide components differed. The core oligosaccharide of B. parapertussis was chemically distinct from the core of B. bronchiseptica which appeared to be structurally and immunologically similar to a core oligosaccharide of B. pertussis.

Structural features involved in the mitogenic activity of Bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice.

Spleen cells from the C3H/HeJ mouse strain cannot be stimulated by many smooth-type lipopolysaccharides (LPSs), and by the main biologically-active region (lipid A) of these molecules. The genetic origin of this defect (expression of the mutant allele Lpsd at the chromosome 4 locus) was established over 20 years ago, but its biochemical nature has remained undefined. Several investigators have noted, however, that some particular LPSs can bypass this defect, and stimulate the proliferation of C3H/HeJ B lymphocytes. In this study we compare the mitogenic activities of the LPSs isolated from a wild strain (1414) and from a mutant 'rough' strain (A100) of Bordetella pertussis. Both LPS-1414 and LPS-A100 were mitogenic for C3H/HeJ spleen cells, but their lipid A fragments were not. This indicates that a carbohydrate structure proximal to lipid A is involved in the mitogenic activity. However, the isolated polysaccharides were not mitogenic. Four sugars are common to both LPS-1414 and LPS-A100: an heptose, and three sugars bearing free amino groups. After removal of these four sugars from the LPSs by nitrous acid treatment, the recovered lipooligosaccharides were not mitogenic in Lpsd spleen cells. The results suggest that substructures present in lipid A and in this group of four sugars are both required for induction of a mitogenic effect in Lpsd splenocytes, whereas lipid A alone can stimulate Lpsn spleen cells.

Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of the glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin.

Due to the formation of micelles, severance of the hydrophilic (poly- or oligosaccharide) and hydrophobic ("Lipid A") domains of bacterial lipopolysaccharides at pH 3.4 or 4.5 and 100 degrees is slow and sometimes does not proceed at all; partially degraded fragments are usually formed. At pH 3.4 (100 degrees) in aqueous 1% sodium dodecylsulphate (SDS), both lipopolysaccharides of the Bordetella pertussis endotoxin are cleaved within 20-30 min, but 80% of the glycosidically bound phosphate present in the hydrophobic domain is lost. Other endotoxins behave similarly. At pH 4.5 (100 degrees) and in the absence of detergent, hydrolysis of the glycosidic bonds of 3-deoxy-D-manno-2-octulosonic acid residues of the B. pertussis endotoxin is negligible but, in aqueous 1% SDS, severance of the two regions of LPS 1 is complete within 1 h (that of LPS-2 requires 3-4 h), and the glycosidically bound phosphate of the isolated hydrophobic region is preserved. Comparison of the rate of acid-catalysed hydrolysis of the glycosidically bound phosphate present in this "isolated Lipid A" preparation with that of 2-deoxy-2-[(3R)-3-hydroxytetradecanamido]-alpha- and -beta-D-glucopyranose 1-phosphates established that the former 1-phosphate was the alpha anomer.

Structure of a hexasaccharide proximal to the hydrophobic region of lipopolysaccharides present in Bordetella pertussis endotoxin preparations.

A branched-chain hexasaccharide containing 3-deoxy-D-manno-oct-2-ulosonic acid was released by detergent-promoted hydrolysis from Bordetella pertussis endotoxin preparations that were first dephosphorylated with aqueous HF and then treated with nitrous acid. Its structure (2) [Formula: See text] was determined by chemical and physical methods. This hexasaccharide is present in all four lipopolysaccharides that make up the B. pertussis strain 1414 (phase 1) endotoxin preparations analysed, and is situated near to the hydrophobic domains. An analogous structure reported previously (ref 7) is erroneous and should be disregarded.

Draft Genome Sequences of Bordetella hinzii and Bordetella trematum.

Bordetella hinzii colonizes the respiratory tracts of poultry but can also infect immunocompromised humans. Bordetella trematum, however, only infects humans, causing ear and wound infections. Here, we present the first draft genome sequences of strains B. hinzii ATCC 51730 and B. trematum CCUG 13902.

Matrix implanted laser desorption ionization (MILDI) combined with ion mobility-mass spectrometry for bio-surface analysis.

The implantation of low velocity massive gold cluster ions allows homogeneous incorporation of a metallic matrix into the near-surface region of rat brain tissues. Subsequent analysis by laser desorption ionization mass spectrometry yields spectra exhibiting molecular ion peaks in the mass range up to 35 kDa similar to those observed by matrix-assisted LDI. Matrix-implanted LDI when combined with ion-mobility preseparation promises to be a useful technique for molecular imaging of biotissues with a laser microprobe.

252Cf plasma desorption mass spectrometry applied to the analysis of underivatized rough-type endotoxin preparations.

Plasma desorption mass spectrometry has recently been used with success to characterize native, underivatized Re- to Rc-type endotoxins in terms of their constituent lipopolysaccharides. The spectra give masses for the major molecular species of lipopolysaccharide present from which their probable compositions could be inferred using the overall composition determined by chemical analyses. Moreover, the relative intensities of the signals are roughly proportional to the abundance of their corresponding molecular species. Native Rc-, Rb-, and Ra-type enterobacterial endotoxins with 5-10 core sugar units have been rendered amenable to plasma-desorption mass spectrometry analysis by improvement in their solubility and the use of cellobiose as an additive. The spectra of four Salmonella and Escherichia endotoxin preparations demonstrated heterogeneity in acylation and phosphorylation. Since these sources of heterogeneity are critical for many biological activities, the spectra underline the need to define the composition of each preparation of endotoxin used in structure-function studies.

Comparison of lipids A of several Salmonella and Escherichia strains by 252Cf plasma desorption mass spectrometry.

Plasma desorption mass spectrometry has recently been used with success to characterize underivatized lipid A preparations: the major molecular species present give signals indicating their masses, from which probable compositions could be inferred by using the overall composition determined by chemical analyses. In the present study, plasma desorption mass spectrometry was used to compare structures in lipid A preparations isolated from several smooth and rough strains of Escherichia and Salmonella species. Preparations isolated from strains of both genera revealed considerable variation in degree of heterogeneity (number of fatty acids and presence or absence of hexadecanoic acid, phosphorylethanolamine, and aminoarabinose). Molecular species usually associated with Salmonella lipid A were found in preparations from Escherichia sp. In addition, preparations from three different batches of lipid A from one strain of Salmonella minnesota showed significant differences in composition. These results demonstrate that preparations used for biological and structural analyses should be defined in terms of their particular molecular constituents and that no generalizations based on analysis of a single preparation should be made.

Do endotoxins devoid of 3-deoxy-D-manno-2-octulosonic acid exist?

After treatment with aqueous, 50% hydrofluoric acid, a well-known dephosphorylating agent, the presence of 3-deoxy-D-manno-2-octulosonic acid (KDO), an essential and characteristic constituent of endotoxins, can be readily demonstrated in reportedly KDO-deficient bacterial lipopolysaccharides.

Direct microextraction and analysis of rough-type lipopolysaccharides by combined thin-layer chromatography and MALDI mass spectrometry.

A rapid method for the microscale extraction of lipopolysaccharides (endotoxins, LPSs) from rough-type Gram-negative bacteria was developed using thin-layer chromatography (TLC) combined with improved conditions for LPS analysis by mass spectrometry. TLC of intact bacteria on silica gel plates in an appropriate solvent selectively extracted and separated their LPS components. The bands of molecular species were scraped from the plates after nondestructive visualization, directly mixed with matrix, and analyzed by laser desorption time-of-flight mass spectrometry. Lipids A and Re-type LPSs were analyzed after transfer to a membrane. Adding citric acid to the matrix gave greatly improved mass spectra. The system allows characterization of bacterial LPS at the microscale level and is equally well applicable to heterogeneous LPS and lipid A preparations (Escherichia coli lipid A and Bordetella lipopolysaccharides were used). The technique provides a rapid determination of the heterogeneity of unmodified preparations and the determination of the molecular weight of each separated component.

Several uses for isobutyric Acid-ammonium hydroxide solvent in endotoxin analysis.

Many steps in the analysis of rough and semirough endotoxins were found to be facilitated by the use of isobutyric acid-ammonium hydroxide solvent.

The specific capsular polysaccharide of Streptococcus pneumoniae type 15A (American type 30).

The specific capsular polysaccharide of Streptococcus pneumoniae type 15A (American type 30) is composed of D-galactose (three parts), D-glucose (one part), 2-acetamido-2-deoxy-D-glucose (one part), phosphate (one part), and glycerol (one part). Hydrolysis, periodate oxidation, methylation, optical rotation, and nuclear magnetic resonance studies showed that the polysaccharide is a high molecular weight linear polymer of a pentasaccharide repeating unit having the structure: (formula: see text)