Structural elucidation of the O-chain of the lipopolysaccharide from Xanthomonas campestris strain 8004

A novel O-specific polysaccharide containing 3-acetamido-3-deoxy-alpha-D-fucose (Fuc3NAc) and D-rhamnose was isolated from the phenol-soluble lipopolysaccharide fraction of the plant associated bacterium Xanthomonas campestris strain 8004. The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a branched trisaccharide repeating unit, as shown below: [formula: see text].     

The structure of the core region of the lipopolysaccharide from Geobacter sulfurreducens

The structure of the core part of the LPS from Geobacter sulfurreducens was analysed. The LPS contained no O-specific polysaccharide (O-side chain) and upon mild hydrolysis gave a core oligosaccharide, which was isolated by gel chromatography. It was studied by chemical methods, NMR and mass spectrometry, and the following structure was proposed. [carbohydrate structure: see text] where Q = 3-O-Me-alpha-L-QuiNAc-(1-->or H (approximately 3:2).     

An improved rapid method for the preparation of D-rhamnose

A method is developed for the preparation of D-rhamnose from an O-polysaccharide (OPS) isolated by mild acid hydrolysis of Azospirillum brasilense SR75 cell mass. After the OPS hydrolysis, D-rhamnose was recovered by gel-permeation chromatography on Toyopearl TSK HW-40 and was crystallized. The sugar activity was demonstrated immunochemically. The advantages of the method are that it expedites and simplifies the extraction of D-rhamnose and increases its yield.     

Structure of the O-polysaccharide of Pseudomonas putida FERM P-18867

The O-polysaccharide of the lipopolysaccharide of Pseudomonas putida FERM P-18867 was found to contain D-mannose and D-rhamnose and have the following structure of the trisaccharide repeating unit:-->2)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-beta-D-Manp-(1-->     

Characterization of the lipopolysaccharide from a wbjE mutant of the serogroup O11 Pseudomonas aeruginosa strain, PA103

The lipopolysaccharide (LPS) of a wbjE mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain consists of both high and low molecular weight (HMW and LMW) LPSs. The HMW LPS consisted exclusively of rhamnan A-band LPS and no B-band LPS was detected in the wbjE mutant. Interestingly, the LMW LPS from the wbjE mutant showed that it contained a variety of oligosaccharides, each with two or three phosphate groups present as mono- or pyrophosphates. These oligosaccharides consisted of the complete core octasaccharide. The GalN residue was present as an N-acetylated residue in all of these oligosaccharides except the tetrasaccharide in which it is present as an N-alanylated residue. None of these oligosaccharides contained either a d- or l-FucpNAc residue. These results are discussed with regard to the role of wbjE in the biosynthesis of P. aeruginosa PA103 B-band LPS.     

Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response

In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents. Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls. Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized. At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae. Injection of Grace's medium, M. lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding. The protein banding patterns of parasitized hosts did not correspond to those of the other treatments. Two parasitoid-induced proteins (38 and 128 kDa) were examined. Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M. lysodeikticus or LPS. Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.     

A Mesorhizobium lipopolysaccharide (LPS) specific lectin (CRL) from the roots of nodulating host plant, Cicer arietinum

A 30 kDa rabbit erythrocyte agglutinating glycoprotein isolated and characterized from the roots of Cicer arietinum and designated as cicer root lectin (CRL). Hemagglutination activity of CRL is strongly inhibited by cell surface LPS of nodulating cicer specific Rhizobium. CRL agglutinates mesorhizobial cells and not Escherichia coli or yeast cells. It binds to immobilized LPS of cicer specific Rhizobium only. The primary structure of CRL as predicted by peptide mass fingerprinting by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) indicated ∼54% amino acid sequence homology with C. arietinum seedling lectin (Accession no. gi/3204123) and ∼26% with C. arietinum (Accession no. gi/110611256), and Pisum sativum (Accession nos. gi/230612, gi/6729956, gi/126148) lectins. These suggested CRL to be a member of vegetative tissue lectin. Circular dichroism analysis indicated that the secondary structure of CRL consists of 48% β-sheets, 26% random coils, and 11% α-helix. CRL has six isoforms of closely associated molecular mass with differential acidic pI of 5.30, 5.20, 5.15, 5.05, 5.00, 4.80. Identity of these isoforms was confirmed from their binding with cicer specific Rhizobium LPS. All the isoforms of CRL are differentially glycosylated as identified by deglycosylation and monosaccharide analysis using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). All these results suggest that unlike other plant lectins CRL is a LPS-binding lectin.     

The linkage between O-specific caryan and core region in the lipopolysaccharide of Burkholderia caryophylli is furnished by a primer monosaccharide

From the lipopolysaccharide (LPS) fraction of the plant-pathogenic bacterium Burkholderia caryophylli, the linkage between O-specific caryan and core region was characterised. The LPS fraction was first treated with 48% aqueous HF at 4 degrees C and successively with 1% acetic acid at 100 degrees C. A main oligosaccharide representing the carbohydrate backbone of the core region and a portion of the caryan (three unit of caryose) was isolated by high-performance anion-exchange chromatography. Compositional and methylation analyses, matrix-assisted laser desorption/ionisation mass spectrometry and 2D NMR spectroscopy identified the structure: [carbohydrate structure: see text]. The above residues are alpha-linked pyranose rings, if not stated otherwise. Hep is L-glycero-D-manno-heptose, Car is 4,8-cyclo-3,9-dideoxy-L-erythro-D-ido-nonose and Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid. This finding indicates that QuiNAc residue is the primer monosaccharide, which connects the core oligosaccharide to caryan O-chain.     

A novel Helicobacter pylori cell-surface polysaccharide

Helicobacter pylori bacteria colonize the gastric mucosa of more than half of the world's human population and its infection may instigate a wide spectrum of gastric diseases in the host. At the moment, there is no vaccine against H. pylori, a microorganism recognized as a category 1 human carcinogen, and treatment is limited to antibiotic management. Pioneering antigenic studies carried out by Penner and co-workers, which employed homologous H. pylori antisera specific for cell-surface lipopolysaccharide (LPS), revealed the presence of six distinct H. pylori serotypes (O1 to O6). Subsequent studies have shown that H. pylori serotype O1 expressed LPS with lengthy O-chain polysaccharide (PS) composed of Lewis blood-group structures ('Lewis O-chains'), serotype O3 LPS produced 'Lewis O-chains' attached to a heptoglycan domain, serotype O4 LPS possessed LPS with glucosylated 'Lewis O-chains' and serotype O6 LPS expressed the heptoglycan domain capped by a short 'Lewis O-chain'. These LPSs were terminated at the reducing-end by a core oligosaccharide and lipid A of conserved structures. With the intent of formulating a multivalent H. pylori LPS-based vaccine, we are studying the structural variability of H. pylori cell-surface glycans. Here, we describe the novel LPS structure produced by H. pylori serotype O2 that differed markedly from the typical H. pylori 'Lewis O-chain' structures, in that its main component was an elongated PS composed of alternating 2-, and 3-monosubstituted alpha-D-Glcp residues [-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]n. These findings revealed the bio-molecular basis for the observed serospecificity of H. pylori serotype O2, and that this unique bacterial PS must be included in the formulation of a multivalent LPS H. pylori vaccine.     

The structure of the carbohydrate backbone of the rough type lipopolysaccharides from Proteus penneri strains 12, 13, 37 and 44

The following structure of the lipid A-core backbone of the rough type lipopolysaccharides (LPS) from Proteus penneri strains 12, 13, 37, and 44 was determined using NMR and mass spectroscopy and chemical analysis of the oligosaccharides obtained by mild-acid hydrolysis, alkaline O,N-deacylation, O-deacylation with hydrazine, and deamination of the LPSs:where K=H, R=PEtN, R(1)=alpha-Hep-(1-->2)-alpha-DDHep, and R(2)=alpha-GalN (strains 12 and 13) or beta-GlcNAc-(1-->4)-alpha-GlcN (strains 37 and 44). LPS from each strain contained several structural variants. LPS from strain 12 contained a variant with R(1)=alpha-DDHep, whereas LPS from strains 13, 37, and 44 contained structures with K=amide of beta-GalA with putrescine or spermidine. The phosphate group at O-1 of the alpha-GlcN residue in the lipid part was partially substituted with Ara4N.     

Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH-I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity.     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.     

Structure and serological characterization of 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid isolated from lipopolysaccharides of Vibrio parahaemolyticus O2 and O-untypable strain KX-V212

Lipopolysaccharides (LPS) of Vibrio parahaemolyticus O2 and O-untypable (OUT) strain (KX-V212) isolated from an individual patient were shown to contain 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid (NonlA), which was readily released from LPS by mild acid hydrolysis. In the present study, we investigated the chemical and serological properties of NonlA isolated from LPS of V. parahaemolyticus O2 and OUT KX-V212. GC-MS and NMR analysis identified the NonlA from LPS of O2 to be 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAcNonlA) and that from LPS of KX-V212 to be 5-acetamido-7-(N-acetyl-D-alanyl)amido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAlaNAcNonlA). In ELISA inhibition analysis, 5NAc7NAcNonlA inhibited the O2 LPS/anti-O2 antiserum system, whereas, 5NAc7NAlaNAcNonlA did not show any inhibitory activity. However, after N-deacylation of 5NAc7NAlaNAcNonlA followed by N-acetylation, the product (5NAc7NAcNonlA) inhibited the O2 LPS/anti-O2 antiserum system to the same extent as that of 5NAc7NAcNonlA obtained from O2 LPS. These results suggest that 5NAc7NAcNonlA might be related to the serological specificity of O2 LPS as one of main epitope(s) involved in O2 LPS.     

The structure of the rough-type lipopolysaccharide from Shewanella oneidensis MR-1, containing 8-amino-8-deoxy-Kdo and an open-chain form of 2-acetamido-2-deoxy-D-galactose

The LPS from Shewanella oneidensis strain MR-1 was analysed by chemical methods and by NMR spectroscopy and mass spectrometry. The LPS contained no polysaccharide O-chain, and its carbohydrate backbone had the following structure: (1S)-GalNAco-(1-->4,6)-alpha-Gal-(1-->6)-alpha-Gal-(1-->3)-alpha-Gal-(1-P-3)-alpha-DDHep-(1-->5)-alpha-8-aminoKdo4R-(2-->6)-beta-GlcN4P-(1-->6)-alpha-GlcN1P, where R is P or EtNPP. There are several novel aspects to this LPS. It contains a novel linking unit between the core polysaccharide and lipid A moieties, namely 8-amino-3,8-dideoxy-D-manno-octulosonic acid (8-aminoKdo) and a residue of 2-acetamido-2-deoxy-D-galactose (N-acetylgalactosamine, GalNAco) in an open-chain form, linked as cyclic acetal to O-4 and O-6 of D-galactopyranose. The structure contains a phosphodiester linkage between the alpha-D-galactopyranose and D-glycero-D-manno-heptose (DDHep) residues.     

Lack of specific binding of labeledRhizobium japonicum lipopolysaccharides to wild soybean (Glycine soja) roots

Summary Lipopolysaccharides (LPS) were extracted from two strains ofRhizobium japonicum (61A76NS and 3I1b110-I). The extracted LPS was purified by gel filtration column chromatography and the amount of 2-keto-3-deoxyoctonate (KDO) was determined. Column purified LPS from both strains were conjugated to rhodamine isothiocyanate on celite to examine binding of this purified, labeled surface component to aseptically grownGlycine soja (wild soybean) seedlings as a basis for symbiotic specificity using fluorescent microscopy. Rhodamine conjugated LPS from both strains ofRhizobium japonicum did not exhibit specific binding to wild soybean seedling roots.Paper no. 8130 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

Preparative separation of isoquinoline alkaloids from Stephania yunnanensis by pH-zone-refining counter-current chromatography

In this paper, five isoquinoline alkaloids were successfully separated from a crude extract of Stephania yunnanensis using pH-zone-refining counter-current chromatography in single-step. With a two-phase solvent system composed of methyl-tert-butyl ether (MtBE)–acetonitrile–water (2:2:3, v/v) where triethylamine (10 mM) was added to the upper organic phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.4 g crude extract, 68.7 mg isocorydine, 78.2 mg corydine, 583.4 mg tetrahydropalmatine, 36.3 mg N-methylasimilobine, and 47.3 mg anonaine were separated with purities over 90%. Their structures were identified by ¹H NMR, ¹³C NMR, ESI-MS data.     

The structure of the core region of the lipopolysaccharide from Shewanella algae BrY, containing 8-amino-3,8-dideoxy-D-manno-oct-2-ulosonic acid

The structure of the carbohydrate backbone of the lipid A-core region of the LPS from Shewanella algae strain BrY was analysed. The LPS was N,O-deacylated to give three products, which were isolated and studied by chemical methods, NMR and mass spectrometry: [Carbohydrate structures: see text]. All monosaccharides except L-rhamnose had the D-configuration. This LPS presents a second example (after S. oneidensis) of the structure with a novel linking unit between the core and lipid A moieties, 8-amino-3,8-dideoxy-D-manno-oct-2-ulosonic acid (8-amino-Kdo).     

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways.     

Tyrosine fluorescence analysis of apolipophorin III-lipopolysaccharide interaction

Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein that binds to lipopolysaccharides (LPS). Polyacrylamide gel electrophoresis analysis demonstrated that apoLp-III from Galleria mellonella associated with various truncated LPS variants, including lipid A. Subsequent binding studies were performed employing the intrinsic tyrosine fluorescence properties of apoLp-III, which is highly quenched in the unbound state. A marked increase in tyrosine fluorescence intensity was observed upon binding to LPS or detoxified LPS, indicating a new microenvironment for Tyr-142. This also implies that the LPS carbohydrate region is involved in LPS binding. Dissociation constants (Kd) measured by apoLp-III titration were estimated at approximately 1 microM. Increasing the ionic strength did not decrease the Kd, neither did LPS phosphate removal. In addition, truncation apoLp-III mutants, lacking two complete helices, were still able to associate with LPS. This indicates that the association of apoLp-III with LPS may not be governed by charge but by hydrophobic interactions.