3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (WaaA) and kdo kinase (KdkA) of Haemophilus influenzae are both required to complement a waaA knockout mutation of Escherichia coli.

The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenzae I69 consists of lipid A and a single 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue substituted with one phosphate at position 4 or 5 (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Zähringer, U. (1988) Eur. J. Biochem. 177, 483-492). The waaA gene encoding the essential LPS-specific Kdo transferase was cloned from this strain, and its nucleotide sequence was identical to H. influenzae DSM11121. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and characterized in vitro to encode a monofunctional Kdo transferase. waaA of H. influenzae could not complement a knockout mutation in the corresponding gene of an Re-type Escherichia coli strain. However, complementation was possible by coexpressing the recombinant waaA together with the LPS-specific Kdo kinase gene (kdkA) of H. influenzae DSM11121 or I69, respectively. The sequences of both kdkA genes were determined and differed in 25 nucleotides, giving rise to six amino acid exchanges between the deduced proteins. Both E. coli strains which expressed waaA and kdkA from H. influenzae synthesized an LPS containing a single Kdo residue that was exclusively phosphorylated at position 4. The structure was determined by nuclear magnetic resonance spectroscopy of deacylated LPS. Therefore, the reaction products of both cloned Kdo kinases represent only one of the two chemical structures synthesized by H. influenzae I69.

Characterization of high affinity monoclonal antibodies specific for chlamydial lipopolysaccharide.

Pathogens belonging to the genus Chlamydia contain lipopolysaccharide with a 3-deoxy-D- manno- oct-2-ulosonic acid (Kdo) trisaccharide of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. This lipopolysaccharide is recognized in a genus-specific pattern by murine monoclonal antibodies (mAbs), S25-23 and S25-2 (both IgG1kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively. The variable domains of these mAbs were reverse transcribed from mRNA which was isolated from hybridomas and cloned as single-chain variable fragments (scFvs) in E.coli TG1. The kinetics of binding of whole antibodies, Fab fragments and scFvs to natural and synthetically modified ligands were determined by surface plasmon resonance (SPR) using synthetic neoglycoconjugates. As examples of an antibody-carbohydrate interaction involving anionic carboxyl groups on the ligand, we report that the affinities of these antibodies are higher than usually observed in carbo-hydrate-protein interactions (K(D)of 10(-3)to 10(-5)M). SPR analy-ses of monovalent Fab and scFv binding to the natural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitting of several measurements at different antibody concentrations) of sensorgram data to a one-to-one interaction model. Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Scatchard analysis of the data gave kinetic and affinity constants that were in good agreement with those obtained by global fitting. The SPR data also showed that while S25-2 bound well to several Kdo disaccharides and carboxyl-reduced Kdo ligands, S25-23 did not. Identification of amino acids in the complementarity determining regions revealed the presence of a large number of positively charged amino acids which were located towards the center of the combining site, thus suggesting a different recognition mechanism than that observed for neutral ligands. The latter mainly involves aromatic amino acids for hydrophobic stacking inter-actions and hydrogen bonds.

The dual role of lipopolysaccharide as effector and target molecule.

Lipopolysaccharides (LPS) are major integral components of the outer membrane of Gram-negative bacteria being exclusively located in its outer leaflet facing the bacterial environment. Chemically they consist in different bacterial strains of a highly variable O-specific chain, a less variable core oligosaccharide, and a lipid component, termed lipid A, with low structural variability. LPS participate in the physiological membrane functions and are, therefore, essential for bacterial growth and viability. They contribute to the low membrane permeability and increase the resistance towards hydrophobic agents. They are also the primary target for the attack of antibacterial drugs and proteins such as components of the host's immune response. When set free LPS elicit, in higher organisms, a broad spectrum of biological activities. They play an important role in the manifestation of Gram-negative infection and are therefore termed endotoxins. Physico-chemical parameters such as the molecular conformation and the charges of the lipid A portion, which is responsible for endotoxin-typical biological activities and is therefore termed the 'endotoxic principle' of LPS, are correlated with the biological activity of chemically different LPS.

The structure of the carbohydrate backbone of the core-lipid A region of the lipopolysaccharide from a clinical isolate of Yersinia enterocolitica O:9.

Yersinia enterocolitica O:9 strain Ruokola/71-c-PhiR1-37-R possesses mainly rough-type lipopolysaccaride (LPS) and smaller amounts of S-form LPS. Structural analysis of the former is reported here. After deacylation of the LPS, the phosphorylated carbohydrate backbone of the inner core-lipid A region could be isolated by using high-performance anion-exchange chromatography. Its structure was determined by means of compositional and methylation analyses and 1H-, 13C-, and 31P-NMR spectroscopy as: [see text] in which L-alpha-D-Hep is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hep is D-glycero-alpha-D-manno-heptopyranose, and Kdo is 3-deoxy-D-manno-oct-2-ulopyranosonic acid. All hexoses are pyranoses.

Isolation and structural analysis of phosphorylated oligosaccharides obtained from Escherichia coli J-5 lipopolysaccharide.

The chemical structure of the phosphorylated lipopolysaccharide (LPS) of Escherichia coli J-5 was investigated because it is of biomedical interest in the context of septic shock, a syndrome often encountered in nosocomial infections with gram-negative pathogens. The successive de-O-acylation and de-N-acylation of J-5 LPS yielded phosphorylated oligosaccharides which represent the complete carbohydrate backbone. Five compounds were separated by high-performance anion-exchange chromatography and analysed by one-dimensional and two-dimensional homonuclear and heteronuclear 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The main product was a nonasaccharide of the structure alpha-D-Glcp-(1-->3)-[alpha-D-GlcpN- (1-->7)-alpha-L,D-Hepp-(1-->7)]-alpha-L,D-Hepp-(1-->3)-alpha -L, D-Hepp-4P-(1-->5)-[alpha-Kdop-(2-->4)]-alpha-Kdop-(2-- >6)-beta-D-GlcpN-4p- (1-->6)-alpha-D-GlcN-1P wherein all sugars are present as D-pyranoses. Hep and Kdo represent L-glycero-D-manno-heptose and 3-deoxy-D-manno-oct-2-ulosonic acid, respectively. In addition, two octasaccharides and two heptasaccharides were isolated that were partial structures of the nonasaccharide. In one octasaccharide the terminal alpha-D-GlcpN was missing and an additional phosphate group linked to O4 of the branched heptose was present, whereas in the other octasaccharide the side-chain Kdo was missing. In both heptasaccharides the side-chain alpha-D-GlcpN-(1-->7)-L-alpha-D-Hepp-disaccharide was absent; they differed in their phosphate substitution. Whereas both heptasaccharides contained two phosphates in the lipid-A backbone (beta-1,6-linked GlcpN-disaccharide at the reducing end) and one phosphate group at O4 of the first heptose, only one of them was additionally substituted with phosphate at O4 of the second heptose.

Structural investigation of the lipopolysaccharide from Acinetobacter haemolyticus strain NCTC 10305 (ATCC 17906, DNA group 4).

The structure of the lipopolysaccharide (LPS) from Acinetobacter haemolyticus strain NCTC 10305 (DNA group 4) was elucidated by means of analytical chemistry, NMR spectroscopy and fast-atom-bombardment mass spectrometry. Several oligosaccharides were obtained after deacylation or successive de-O-acylation, dephosphorylation, reduction, and de-N-acylation of LPS. In the major fraction of the LPS, the core is attached to the lipid A through D-glycero-D-talo-2-octulopyranosonic acid (Ko), whereas in a minor fraction (<20%) Ko is replaced by 3-deoxy-D-manno-octulopyranosonic acid (Kdo). The structures of the phosphorylated carbohydrate backbones of these LPS fractions are [structure: see text] with Dha = 3-deoxy-D-lyxo-2-heptulosaric acid, Sug = sugar and is Ko in a major fraction and Kdo in a minor fraction. All sugar residues have the D-configuration and are present in the pyranose form. Mass spectrometry of de-O-acylated LPS revealed the presence of an additional hexose residue in minor amounts, the position and nature of which could not be identified.

Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution.

The chromosomal genes rfaC and rfaF of Escherichia coli W3110 were inactivated by allelic-replacement mutagenesis to generate a defined strain lacking both heptosyltransferases which catalyze in lipopolysaccharide (LPS) biosynthesis the transfer of the first two L-glycero-D-manno-heptose (Hep) residues to 3-deoxy-D-manno-2-octulosonic acid (Kdo). The LPS of the mutant was isolated and its chemical structure was investigated by compositional analysis and nuclear magnetic resonance spectroscopy of isolated, deacylated oligosaccharide phosphates. The basic structure was a tetrasaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)-alpha-D- GlcN1P which in LPS was substituted at position 07 of Kdo II by 2-aminoethanol phosphate in non-stoichiometric amounts. 2-Aminoethanol was cleaved during deacylation of the LPS by successive hydrazinolysis and KOH treatment and, in addition, phosphate migration from 07 to 08 of Kdo II occurred. Thus, the oligosaccharides alpha-Kdo7P-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)- alpha-D-GlcN1P and alpha-Kdo8P-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)- alpha-D-GlcN1P could be isolated. KOH treatment of the two trisphosphates and authentic methyl 3-deoxy-D-manno-octulopyranoside 7-(2-acetamidoethyl phosphate) proved that phosphate migration only took place when the phosphate group was substituted with 2-aminoethanol. Complementation studies with plasmid-encoded rfaC and rfaF genes revealed that the mutant strain can be used in combination with LPS-specific antibodies for the cloning and characterization of heptosytransferases which glycosylate Kdo residues of the inner core region of LPS.

Chemical structure of the core region of Escherichia coli J-5 lipopolysaccharide.

The lipopolysaccharide of Escherichia coli J-5 was sequentially de-O-acylated, dephosphorylated, reduced, de-N-acylated, and N-acetylated. The products were separated by high-performance anion-exchange chromatography into a nonasaccharide (1), two octasaccharides (2, 3), and a heptasaccharide (4). Compositional analysis, methylation analysis, and NMR spectroscopy revealed the structures of the products as: alpha-D-GlcpNAc-(1-7)-L-alpha-D-Hepp-(1-7)-[alpha-D-Glcp-(1-3)-]-L -alpha-D- Hepp-(1-3)-R1, (1) L-alpha-D-Hepp-(1-7)-[alpha-D-Glcp-(1-3)-]-L-alpha-D-Hepp-(1 -3)-R1, (2) alpha-D-GlcpNAc-(1-7)-L-alpha-D-Hepp-(1-7)-[alpha-D-Glcp-(1-3)-]-L -alpha-D- Hepp-(1-3)-R2, (3) alpha-D-Glcp-(1-3)-L-alpha-D-Hepp-(1-3)-R1, (4) in which 1R is L-alpha-D-Hepp-(1-5)-[alpha-Kdop-(2-4)-]-alpha-Kdop-(2 -6)- beta-D-GlcpNAc-(1-6)-D-GlcN-Acol, and 2R is L-alpha-D-Hepp-(1-5)-alpha-Kdop-(2-6)-beta-D-GlcpNAc-(1-6 )-D- GlcNAcol (LD-Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-octulopyranosonic acid; GlcNAcol, 2-acetamido-2-deoxy-glucitol). Fast-atom-bombardment mass spectrometry of de-O-acylated and dephosphorylated lipopolysaccharide showed that the isolated oligosaccharides represented the complete carbohydrate moiety of the lipopolysaccharide, and indicated that the non-reducing terminal D-GlcN residue in lipopolysaccharide was present as the free base.

Chemical structure of the lipid A of Escherichia coli J-5.

The lipopolysaccharide, and particularly its lipid A moiety, of the J-5 mutant of Escherichia coli O111 plays a central role in studies on potential induction of cross-reactive and cross-protective antibodies, however, its chemical and antigenic structure was hitherto unknown. Here, the chemical structure of the J-5 lipid A is reported. It is composed of the bisphosphorylated disaccharide beta-D-GlcpN-4-P-(1-6)-alpha-D-GlcpN-1-P which carries four residues of 3-hydroxytetradecanoic acid, one each at positions 2, 3, 2', and 3'. The hydroxyl groups of the acyl residues at 2' and 3' are esterified with dodecanoic and tetradecanoic acid, respectively. The hydroxyl group at C-6' functions in the lipopolysaccharide as the attachment site of the core oligosaccharide. Furthermore, a new method to isolate the hydrophilic backbone, i.e. the 1,4'-bisphosphorylated glucosamine disaccharide, and its structural analysis by 1H-, 13C-, and 31P-NMR spectroscopy, are described, leading to a new and easier strategy in structural analysis of lipid A from bacterial lipopolysaccharides.