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LPS is a fundamental constituent of the outer membrane of all Gram-negative bacteria, and the lipid A domain plays a central role in the induction of inflammatory responses. We identified genes of the Neisseria gonorrhoeae lipid A biosynthetic pathway by searching the complete gonococcal genome sequence with sequences of known enzymes from other species. The lpxLll gene was disrupted by an insertion-deletion in an attenuated aroA mutant of the gonococcal strain MS11. Lipopolysaccharide (LPS) and lipid A analysis demonstrated that the lpxLII mutant had synthesized an altered LPS molecule lacking a single lauric fatty acid residue in the GIcN II of the lipid A backbone. LPS of the IpxLll mutant had a markedly reduced ability to induce the proinflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-8 from human macrophages and lL-8 from polymorphonuclear cells. This study demonstrates that the lpxLII gene in gonococci encodes for a late-functioning lauroyl acyl transferase that adds a lauric acid at position 2' in the lipid A backbone. The presence of lauric acid at such a position appears to be crucial for the induction of full inflammatory responses by N. gonorrhoeae LPS. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide (LPS) expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis patients lacks the O-polysaccharide chain but the degree to which the rest of the molecule changes has not been determined. We analyzed, for the first time, the core structure of an LPS from a rough, cystic fibrosis isolate of P. aeruginosa. The products of mild acid hydrolysis and strong alkaline degradation of the LPS were studied by ESI MS, MALDI MS, and NMR spectroscopy. The following structure was determined for the highest-phosphorylated core-lipid A backbone oligosaccharide isolated after alkaline deacylation of the LPS: &figf7; where Kdo and Hep are 3-deoxy-d-manno-octulosonic acid and l-glycero-d-manno-heptose, respectively; all sugars are in the pyranose form and have the d configuration unless stated otherwise. The outer core region occurs as two isomeric glycoforms differing in the position of rhamnose (Rha). The inner core region carries four phosphorylation sites at two Hep residues, HepI being predominantly bisphosphorylated and HepII monophosphorylated. In the intact LPS, both Hep residues carry monophosphate and diphosphate groups in nonstoichiometric quantities, GalN is N-acylated by an l-alanyl group, HepII is 7-O-carbamoylated, and the outer core region is nonstoichiometrically O-acetylated at four sites. Therefore, the switch to the LPS-rough phenotype in cystic fibrosis isolates of P. aeruginosa is not accompanied by losses of core monosaccharide, phosphate or acyl components. The exact positions of the O-acetyl groups and the role of the previously undescribed O-acetylation in the LPS core of P. aeruginosa remain to be determined. [ABSTRACT FROM AUTHOR]

Ceramide is a pivotal molecule in signal transductionand an essential structural component of the epidermal permeability barrier.The epidermis is marked by a high concentration of ceramide andby a unique spectrum of ceramide species: Besides the two ceramidestructures commonly found in mammalian tissue, N-acylsphingosineand N-2-hydroxyacyl-sphingosine, six additionalceramides differing in the grade of hydroxylation of either thesphingosine base or the fatty acid have been identified in the epidermis.Here we report on the characterization of an IgM-enriched polyclonal mouseserum against ceramide. In dot blot assays with purified epidermallipids the antiserum bound to a similar extent to N-acyl-sphingosine(ceramide 2), N-acyl-4-hydroxysphinganine (ceramide3), and N-(2-hydroxyacyl)-sphingosine (ceramide5), whereas no specific reaction was detected with glycosylceramides,sphingomyelin, free sphingosine, phospholipids, or cholesterol.In contrast, a monoclonal IgM antibody, also claimed to be specificfor ceramide, was shown to bind specifically to sphingomyelin andtherefore was not further investigated. In thin-layer chromatography immunostainingwith purified lipids a strong and highly reproducible reaction ofthe antiserum with ceramide 2 and ceramide 5 was observed, whereasthe reaction with ceramide 1 and ceramide 3 was weaker and morevariable. Ceramide 2 and ceramide 5 were detected in the nanomolar rangeat serum dilutions of up to 1:100 by dot blot and thin-layer immunostaining.In thin-layer chromatography immunostaining of crude lipid extractsfrom human epidermis, the antiserum also reacted with N-(2-hydroxyacyl)-4-hydroxysphinganine(ceramide 6) and N-(2-hydroxyacyl)-6-hydroxysphingosine(ceramide 7). Furthermore, the suitability of the antiserum forthe detection of endogenous ceramide by immunolight microscopy wasdemonstrated on cryoprocessed human skin tissue. Double immunofluorescencelabeling experiments with the anti-ceramide antiserum and the recentlydescribed anti-glucosylceramide antiserum (Bradeet al., 2000, Glycobiology 10, 629) showed that both lipids are concentratedin separate epidermal sites. Whereas anti-ceramide stained the dermaland basal epidermal cells as well as the corneocytes, anti-glucosylceramidestaining was concentrated in the stratum granulosum. In conclusion,the specificity and sensitivity of the reagent will enable studieson the subcellular distribution and biological functions of endogenousceramide. [ABSTRACT FROM PUBLISHER]

We here report on the structural analysis of a novel tetra-acyl lipid A (LAtetra) isolated from Escherichia coli deep rough (Re)-mutant strain F515. In addition to the biologically active hexa-acyl E. coli-type lipid A (compound 506), this incompletely acylated lipid A was found to be also present in the native LPS. Its structure was studied without further derivatisation by chemical analysis, matrix-assisted laser desorption/ionization mass spectrometry, and one- and two-dimensional 1H- and 13C-NMR spectroscopy. It was found to be structurally distinct from the tetraacyl lipid A biosynthetic precursor Ia (compound 406) in lacking the primary (R)-3-hydroxytetradecanoic acid 14:0(3-OH) in position 3′ ester-linked to the `non-reducing' glucosamine (GlcN II). The hydroxyl group at the (R)-3-hydroxytetradecanoic acid attached to position 2′ of GlcN II was found to be substituted by dodecanoic acid (12:0), thus forming a dodecanoyloxytetradecanoyl residue 14:0[3- O(12:0)]. The acylation pattern at the `reducing' GlcN I was identical to that of compound 406 in having two primary (R)-3-hydroxy tetradecanoic acid residues [14:0(3-OH)] attached to positions 3 (ester-linked) and 2 (amide-linked), respectively. In human mononuclear cells (hMNC) the new LAtetra antagonized LPS-induced release of interleukine-1 (IL-1), interleukine-6 (IL-6), and tumor necrosis factor (TNF) in a dose-dependant manner with identical antagonistic potency as compared with compound 406. Also like compound 406, it was found to be an agonist in murine macrophage-like J774.1 cells. [ABSTRACT FROM PUBLISHER]

Examines the structure of the lipopolysaccharide from the gram-negative bacteria, Chlamydophila psittaci strain 6BC. Chemical composition; Molar ratio of organic bound phosphate and fatty acids; Distribution of fatty acids in lipid A.

The biosynthesis of lipid A component was shown to be defective in a temperature-sensitive firA mutant of Escherichia coli. Cells were biosynthetically labelled with [14C]acetate rand incorporation of radioactivity into the glycerophospholipid compared to lipid A fractions was measured. The lipid A/glycerophospholipid biosynthesis ratio of the firA mutant at 37°C was approximately 50%, and at the nonpermissive temperature of 42°C was less than 20% of that observed in the corresponding wild-type strain. Analysis of radiolabelled lipid A 4′-monophosphate derivatives and glycerophospholipids by thin-layer chromatography revealed that the firA mutant at 42 °C elaborated an altered lipid A, and its phosphatidylglycerol content was low. The chemical, composition of the extracted lipopolysaccharides differed significantly between the firA and the wild-type strain only in the proportion of hexadecanoic acid, which was minimal in the wild type grown at 37°C and 42°C and in firA lipopolysaccharide grown at 37°C. In the firA mutant lipopolysaccharide produced at 42°C, hexadecanoic acid was present in approximately every third molecule, attached to the hydroxyl group of the amide-linked (R)-3-hydroxytetradecanoic acid at the reducing glucosamine of lipid A. Inspection of dephosphorylated e lipid A preparations by laser-desorption mass spectrometry, confirmed that significant amounts of heptaacyl lipid A was elaborated by the firA strain grown at 42°C. [ABSTRACT FROM AUTHOR]

The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd¯/b¹) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a β-(1′r,6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4′ of the non-reducing one. Four molecules of (R)-3-hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2, 2′ and 3, 3′, respectively). Laser-desorption mass spectrometry showed that both 3-hydroxytetradecanoic acids linked to the non-reducing glucosamine carry tetradecanoic acid at their 3-hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A. The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583– 587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas-Uquid chromatography/mass spectrometry) also revealed dOclA 5-phosphate. Since a cyclic 4,5-phosphate could be ruled out by 31P-NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4- and 5-phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C-6′ of the nonreducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by ¹HNMR and 13C-NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure: dOcl Apα(2″–6′)GlcNpβ6GlcNpαP. [ABSTRACT FROM AUTHOR]

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12°C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetradecanoic acid at GlcN II is positionally replaced by Δ9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to Glecn II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to Glen I are free or only partially substituted. [ABSTRACT FROM AUTHOR]

Presents a study which explain the chemical structure of free lipid A isolated from rough- and smooth-form lipopolysaccharides of the human gastroduodenal pathogen Helicobacter pylori. Information on Helicobacter pylori; Chemical composition of lipid A; Structural analysis of the lipid A; Results of this study.

Presents information on a study which hypothesized that Yersinia (Y) pseudotuberculosis and Yersinia pestis by comparison are more resistant to bactericidal cationic peptides than Yersinia enterocolitica. Analysis of polycation interaction with lipopolysaccharide (LPS); Information on the polycation binding of LPS of cells grown at 37 degrees Celsius; How pathogenic Yersinia pseudotuberculosis and Yersinia pestis causes intestinal infections.

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero- d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4′ by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3′ were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo. [ABSTRACT FROM AUTHOR]

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy- d-manno-octulosonic acid region of lipopolysaccharides. Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy- d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue. The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy- d-manno-octulosonic acid and/or other polar substituents. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides isolated from the polymyxin-resistant Klebsiella pneumoniae O3 mutant OM-5 and its polymyxin-sensitive parent LEN-1 were analyzed for chemical composition, and their lipid A portions were structurally characterized. The lipopolysaccharide of OM-5 contained approximately five times more 4-amino-4-deoxy-L-arabinopyranose than that of LEN-1. Other saccharide and phosphate components exhibited no significant differences. Structural characterization, including analyses by phosphorus magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry, revealed a novel type of lipid A. In the OM-5 lipopolysaccharide, both phosphates of lipid A were almost totally present as phosphodiesters with 4-amino-4-deoxy-L-arabinopyranose. In the sensitive-type LEN-1 lipid A, the extent of this substitution was much lower, especially in the glycosidically linked phosphate. Phosphate in these K. pneumoniae lipopolysaccharides was almost exclusively found in lipid A. These results show that cationic substituents of phosphates of lipid A play a decisive role in determining polymyxin reactivity. OM-5 was also found to contain a large proportion of heptaacyl lipid A, which represented only a small fraction of lipid A in LEN-1. [ABSTRACT FROM AUTHOR]

The chemical structure of the lipid A components of smooth-type lipopolysaccharides isolated from the type strains of strictly anaerobic beer-spoilage bacteria Pectinatus cerevisiiphilus and Pectinatus frisingensis were analyzed. The hydrophilic backbone of lipid A was shown, by controlled degradation of lipopolysaccharide combined with chemical assays and [sup31]P-NMR spectroscopy, to consist of the common β 1-6-linked disaccharide of pyranosidic 2-deoxy-glucosamine (GlcN), phosphorylated at the glycosidic position and at position 4'. In de-O-acylated lipopolysaccharide, the latter phosphate was shown to be quantitatively substituted with 4-amino-4-deoxyarabinose, whereas the glycosidically linked phosphate was present as a monoester. Laser-desorption mass spectrometry of free dephosphorylated lipid A revealed that the distal (non-reducing) GlcN was substituted at positions 2' and 3' with (R)-3-(undecanoyloxy)tridecanoic acid, whereas the reducing GlcN carried two unsubstituted (R)-3-hydroxytetradecanoic acids at positions 2 and 3. The lipid A of both Pectinatus species were thus of the asymmetric hexaacyl type. The linkage of lipid A to polysaccharide in the lipopolysaccharide was relatively resistant to acid-catalyzed hydrolysis, enabling the preparation of a dephosphorylated and deacylated saccharide backbone. Methylation analysis of the backbone revealed that position 6' of the distal GlcN of lipid A was the attachment site of the polysaccharide. Despite the quantitative substitution of the lipid A 4'-phosphate by 4-amino-4-deoxyarabinose, which theoretically should render the bacteria resistant to polymyxin, P. cerevisiiphilus was shown to be susceptible to this antibiotic. P. cerevisiiphilus was, however, also susceptible to vancomycin and bacitracin, indicating that the outer membrane of this bacterium does not act as an effective permeability barrier. [ABSTRACT FROM AUTHOR]

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. Its is composed of the bisphosphorylated disaccharide β-D-GlcpN-4-P-(1-6)-α-D-GlcpN-t-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. [ABSTRACT FROM AUTHOR]

The structure of the lipid A component of lipopolysaccharides isolated from two wild-type strains (Fisher 2 and 7) and one rough mutant (PAC 605) of Pseudomonas aeruginosa was investigated using chemical analysis, methylation analysis, combined gas-liquid chromatography/mass spectrometry, laser-desorption mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of a pyranosidic β1,6-linked D- glucosamine disaccharide [β-D-GlcpN-(1 → 6)-D-GlcpN], phosphorylated in positions 4′ and 1. Position 6′ of the β-D-GlcpN-(1 → 6)-D-GlcpN disaccharide was identified as the attachment site of the core oligosaccharide and the hydroxyl group at C-4 was not substituted. Lipid A of the three P. aeruginosa strains expressed heterogeneity with regard to the degree of acylation: a hexaacyl as well as a pentaacyl component were structurally characterized. The hexaacyl lipid A contains two amide-bound 3-O-acylated (R)-3-hydroxydodecanoic acid groups [12:0(3-OH)] at positions 2 and 2′ of the GlcN dissacharide and two ester-bound (R)-3-hydroxydecanoic acid groups [10:0(3- OH)] at positions 3 and 3′. The pentaacyl species, which represents the major lipid A component, lacks one 10:0(3- OH) residue, the hydroxyl group in position 3 of the reducing GlcN residue being free. In both hexa- and penta- acyl lipid A the 3-hydroxyl group of the two amide-linked 12:0(3-OH) residues are acylated by either dodecanoic (12:0) or (S)-2-hydroxydodecanoic acid [12:0(2-OH)], the lipid A species with two 12:0(2-OH) residues, however, being absent. The presence of only five acyl residues in the major lipid A fraction may account for the low endotoxic activity observed with P. aeruginosa lipopolysaccharide. [ABSTRACT FROM AUTHOR]

The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd¯/b¹) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a β-(1′r,6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4′ of the non-reducing one. Four molecules of (R)-3-hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2, 2′ and 3, 3′, respectively). Laser-desorption mass spectrometry showed that both 3-hydroxytetradecanoic acids linked to the non-reducing glucosamine carry tetradecanoic acid at their 3-hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A. The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583– 587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas-Uquid chromatography/mass spectrometry) also revealed dOclA 5-phosphate. Since a cyclic 4,5-phosphate could be ruled out by 31P-NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4- and 5-phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C-6′ of the nonreducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by ¹HNMR and 13C-NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure: dOcl Apα(2″–6′)GlcNpβ6GlcNpαP. [ABSTRACT FROM AUTHOR]

It has previously been demonstrated [Eur. J. Biochem. 124, 191–198 (1982) and 137, 15–22 (1983)] that the lipid A component of Salmnonella and Proteus lipopolysaccharides contains amide-linked (R)-3-acyloxyacyl residues. In the present study lipid A of other gram-negative bacteria was analysed for the presence of amide- bound 3-acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested [Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteriodes fragilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide-linked (R)-3-acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A. The composition of 3-acyloxyacyl groups differed considerably among different bacteria. As amide-bound (R)-3-hydroxy fatty acids straight chain and isobranched acyl groups with 10–17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3-hydroxyl group of 3-hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10–17 carbon atoms as well as (S)-2-hydroxy fatty acids with 12 carbon atoms. In some cases, using laser desorption mass spectrometry, the distribution of 3-acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated. [ABSTRACT FROM AUTHOR]

The structure of the polysaccharide O-chain of the lipopolysaccharide isolated from the sequenced strain Chromobacterium violaceum ATCC 12472 (NCTC 9757) was investigated by chemical and NMR analyses, and concluded to be -4-α-Leg5Ac7Ala-4-β- d -ManNAlaA3OAc-3-α- d -GlcNAc-where Leg5Ac7Ala indicates 5-acetamido-7-alanylamido-3,5,7,9-tetradeoxy- d - glycero - d - galacto -non-2-ulopyranosonic acid and ManNAlaA3OAc 3- O -acetyl-2-alanylamido-2-deoxymannopyranuronic acid. The structure of the core with one repeating unit of the polysaccharide attached was also analyzed, and it was found that the O-chain polysaccharide is linked to the core via β-Glc p NAc, as opposite to α-Glc p NAc inside the O-chain. Image 1 • Chromobacterium violaceum is a Gram-negative coccobacillus, living in soil and water in tropical regions. • C. violaceum can cause fatal infections. • Structure of C. violaceum ATCC 12472 O-antigen and part of the LPS core is presented here. [ABSTRACT FROM AUTHOR]

The rough-type lipopolysaccharide (LPS) of the plague pathogen, Yersinia pestis, was studied after mild-acid and strong-alkaline degradations by chemical analyses, NMR spectroscopy and electrospray-ionization mass spectrometry, and the following structure of the core region was determined:where l-α-d-Hep stands for l-glycero-α-d-manno-heptose, Sug1 for either 3-deoxy-α-d-manno-oct-2-ulosonic acid (α-Kdo) or d-glycero-α-d-talo-oct-2-ulosonic acid (α-Ko), and Sug2 for either β-d-galactose or d-glycero-α-d-manno-heptose. A minority of the LPS molecules lacks GlcNAc. [Copyright &y& Elsevier]

Background Peanut is one of the most hazardous sources of food allergens. Unknown allergens are still hidden in the complex lipophilic matrix. These allergens need to be discovered to allow estimation of the allergenic risk for patients with peanut allergy and to further improve diagnostic measures. Objective We performed detection, isolation, and characterization of novel peanut allergens from lipophilic peanut extract. Methods Extraction of roasted peanuts were performed under defined extraction conditions and examined by means of 2-dimensional PAGE. Subsequently, chromatographic methods were adapted to isolate low-molecular-weight components. Proteins were studied by using SDS-PAGE and immunoblotting with sera from patients with peanut allergy. For allergen identification protein sequencing, homology search and mass spectrometry were applied. Functional characterization for allergenicity was performed by using the basophil activation assay and for antimicrobial activity by using inhibition assays of different bacteria and fungi. Results IgE-reactive proteins of 12, 11, and 10 kDa were first detected after chloroform/methanol extraction in the flow through of hydrophobic interaction chromatography. The proteins were able to activate basophils of patients with peanut allergy. N -terminal sequencing and homology search in the expressed sequence tag database identified the allergens as peanut defensins, which was confirmed by using mass spectrometry. On microbial cell cultures, the peanut defensins showed inhibitory effects on the mold strains of the genera Cladosporium and Alternaria but none on bacteria. Conclusions We identified defensins as novel peanut allergens (Ara h 12 and Ara h 13) that react in particular with IgE of patients with severe peanut allergy. Their antimicrobial activity is solely antifungal. [ABSTRACT FROM AUTHOR]

Psychrobacter arcticus 273-4 is a Gram-negative bacterium isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in the Kolyma region in Siberia. The survival strategies adopted to live at subzero temperatures include all the outer membrane molecules. A strategic involvement in the well-known enhancement of cellular membrane fluidity is attributable to the lipopolysaccharides (LPSs). These molecules covering about the 75% of cellular surface contribute to cold adaptation through structural modifications in their portions. In this work, we elucidated the exact structure of lipid A moiety obtained from the lipopolysaccharide of P. arcticus grown at 4 °C, to mimic the response to the real environment temperatures. The lipid A was obtained from the LPS by mild acid hydrolysis. The lipid A and its partially deacylated derivatives were exhaustively characterized by chemical analysis and by means of ESI Q-Orbitrap mass spectrometry. Moreover, biological assays indicated that P. arcticus 273-4 lipid A may behave as a weak TLR4 agonist. [ABSTRACT FROM AUTHOR]

Erwinia amylovora (E. amylovora) is the first bacterial plant pathogen described and demonstrated to cause fire blight, a devastating plant disease affecting a wide range of species including a wide variety of Rosaceae. In this study, we reported the lipopolysaccharide (LPS) core structure from E. amylovora strain CFBP1430, the first one for an E. amylovora highly pathogenic strain. The chemical characterization was performed on the mutants waaL (lacking only the O-antigen LPS with a complete LPS-core), wabH and wabG (outer-LPS core mutants). The LPSs were isolated from dry cells and analyzed by means of chemical and spectroscopic methods. In particular, they were subjected to a mild acid hydrolysis and/or a hydrazinolysis and investigated in detail by one and two dimensional Nuclear Magnetic Resonance (NMR) spectroscopy and ElectroSpray Ionization Fourier Transform-Ion Cyclotron Resonance (ESI FT-ICR) mass spectrometry. [ABSTRACT FROM AUTHOR]