Hydrolysis Of Lipopolysaccharide Research Articles

Characterization of a Model Bioreactive Membrane for the Simultaneous Separation and Hydrolysis of Lipopolysaccharides

The design of functionalized selective layers to develop novel membranes is essential to provide potential solutions that meet the continuously growing demand of providing safe water. Herein, we present the interfacial response of an enzymatic thin-film composite (E-TFC) membrane displaying dual functionality and fabricated as a proof-of-concept for both efficient lipopolysaccharide (LPS) separation and ester bond hydrolysis. The enzymatic membrane model was constructed by employing lipase b from Candida antarctica (CALB) covalently coupled via chemically activated bisepoxide groups onto the surface of the di-block copolymer polystyrene-b-poly(4-vinyl pyridine) (PS-b-P4VP) layer. Our results show a complete rejection of size-excluded LPS molecules when using the fabricated E-TFC membrane in a forward osmosis (FO) application. Moreover, the immobilized enzyme was able to retain 97% of its enzymatic activity when using 4-nitrophenyl acetate (pNPA) and up to 74% to liberate free fatty acids from LPS molecules within the feed side of the FO system. This work provides fundamental insights into new emerging functional biomaterials that find applications in hybrid catalytic filtration processes that also selectively remove LPS molecules from water sources.

Development and Validation of a Procedure for Authenticity Verification of Modified Lipopolysaccharides of Shigella flexneri Subtypes 1b, 2a, 3a, 6, and Y by 13C NMR Spectroscopy

Clinically applicable lipopolysaccharides (LPSs) from Shigella flexneri, lipid A of which contains at most three higher fatty-acid residues, are promising candidates for creating vaccines against it. NMR studies of modified LPS are practically unreported. Most publications in this field are devoted to studying the polysaccharide structure of native LPS, i.e., O-specific polysaccharide (OPS) that can be isolated after mild hydrolysis of LPS and separation of lipid A. The present article describes the development of an NMR procedure for authenticity verification of modified LPS, i.e., vaccine polysaccharide components. Characteristic 13C resonances in the region of anomeric C atoms are identified. The confidence intervals of their chemical shifts are determined. The procedure is demonstrated to be specific for modified LPS subtypes 1b, 2a, 3a, Y, and 6 from S. flexneri.

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.

The Structure of Uncommon Lipid A from the Marine Bacterium Marinomonas communis ATCC 27118T

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118T with 1% AcOH. Using chemical analysis and ID and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be beta-(1',6)-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-[(R)-3-hydroxydecanoyloxy)]decanoic acid, and (R)-3-hydroxydecanoic acid at the C2, C2' and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M. communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.

Structure of the O-Specific Polysaccharide of Hafnia Alvei 23 Having an Oligosaccharide-Phosphate Repeating Unit

ABSTRACT Lipopolysaccharide (LPS) of Hafnia alvei 23 has an acid-labile O-specific polysaccharide (OPS) with a pentasaccharide-phosphate repeating unit containing D-Glc1P, D-GlcNAc, L-Fuc, 6-deoxy-D-talose (D-6dTal), 4-acetamido-4,6-dideoxy-D-glucose (D-Qui4NAc), and an O-acetyl group. A partially degraded OPS was obtained by hydrolysis of LPS with 0.25 M sodium acetate in aqueous 0.5% acetic acid. Fractionation of LPS on Sephadex G-200 in DOC buffer allowed isolation of long-chain LPS species which, together with OPS, were studied by methylation analysis, chemical degradations (O-deacetylation, dephosphorylation with 48% hydrofluoric acid, Smith degradation), and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and H-detected 1H,13C heteronuclear single-quantum coherence (HSQC) experiments. The following structure of the repeating unit of OPS was established:

Structural and serological characterisation of the O-specific polysaccharide from lipopolysaccharide of Acinetobacter calcoaceticus strain 7 (DNA group 1).

S-form lipopolysaccharide was isolated by phenol/water extraction from a strain of Acinetobacter calcoaceticus (DNA group 1 ). The structure of the O-antigenic polysaccharide was determined by compositional analysis and NMR spectroscopy of the de-O-acylated lipopolysaccharide. The isolated polysaccharide obtained after hydrolysis of lipopolysaccharide in 0.01 M trifluoroacetic acid has the following structure: [STRUCTURE IN TEXT] in which Pyr is pyruvate. The O-acetyl substitution of D-Gal was non-stoichiometric. The O-antigen was specifically recognised in western blots by polyclonal rabbit antisera.

Probing the microheterogeneity of O-specific chains from Yersinia ruckeri using capillary zone electrophoresis/electrospray mass spectrometry.

The analysis of underivatized oligosaccharides arising from mild acid hydrolysis of endotoxins from Yersinia ruckeri serotype O2 was achieved using on-line capillary zone electrophoresis-electrospray mass spectrometry (CZE-ESMS). This technique provided unparalleled resolution of the different glycans obtained from purified fractions of the native endotoxins or from hydrolysis of lipopolysaccharides from Y. ruckeri. Electrophoretic conditions enabling the separation of anionic and cationic analytes were developed to determine possible sites of heterogeneity on either the core or the O-chain glycans. Structural characterization of underivatized oligosaccharides identified in the ion electropherograms was achieved using tandem mass spectrometry under low-collision energy conditions.

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.

Electrospray mass spectrometry for characterization of lipid a fromEnterobacter agglomerans

Negative ion and positive ion electrospray mass spectrometry have been employed to characterize the lipid A mixture produced by hydrolysis of lipopolysaccharides from Enterobacter agglomerans, a Gram-negative bacterium commonly found in field cotton. Neutral monophosphoryl lipid A molecules form stable anions via deprotonation, but adduct formation via cation attachment occurs in low yield. Dephosphorylated lipid A molecules, on the other hand, readily form adducts with Na+, whereas deprotonation occurs in low yield. The mass spectra of lipid A produced by E. agglomerans reveal the presence of lipid A ions which differ in the nature of attached fatty acid side chains. At least two heptaacyl forms of lipid A are present, one of which has a structure which appears to be the same as the structure of heptaacyl lipid A produced by Salmonella minnesota. The second structure differs only by the nature of the side chain at position 3' of the disaccharide backbone where a hydroxymyristoyloxymyristoyl group replaces the myristoyloxymyristoyl substituent. Collisionally activated dissociations prior to mass analysis enable the identification of fragment ions which can be distinguished from at least eight intact deprotonated molecules present in crude lipid A.

Dissociation between mitogenicity and immunogenicity of TNP- lipopolysaccharide, a T-independent antigen

Two models have been proposed to explain triggering of B cells by so-called "T-independent antigen." Feldmann and Basten (1) proposed that the interaction of multiple repeating determinants on polymeric antigens with specific Ig receptors on the B-cell surface is sufficient to provide the signals for division of these cells and differentiation to antibody-forming cells. In contrast, coutinho et al. (2, and see review, 3) have claimed that there is only one signal, a mitogenic signal, receptors acting merely as passive focusing devices to localize the antigen on specific cells where it delivers a mitogenic signal resulting in differentiation to an antibody-producing cell. This model rests primarily on the demonstration that at high concentration all T-independent antigens they have tested are mitogenic for B cells (4-6). Compatible with this hypothesis are the observations that hydrolysis of lipopolysaccharide (LPS) to remove the ester- linked fatty acids of the mitogenic lipid A component abrogates its mitogenic (7,8) activity as well as its ability, when substituted with the TNP hapten, to induce a T-independent anti- TNP response (9). However, alkali treatment of LPS, although not changing its antigenic component (8), may also modify the molecule physically or chemically which could account for loss of immunogenic properties (10). We therefore investigated other reagents which interact with LPS in a more chemically defined manner in an effort to clarify the relationship between the mitogenic and immunogenic properties of this molecule. Polymyxin B (PB) is one of a family of cyclic peptide antibiotics which are bactericidal for most gram-negative bacteria. It prevents the lethal endotoxic activity of LPS (11, 12) and changes the physical structure of LPS (13). We report here that low doses of PB added to cultures of mouse spleen cells inhibit the mitogenic activity of TNP-LPS, a T- independent antigen, and native LPS, but do not suppress the immune response to TNP-LPS. PB interacts with TNP-LPS and LPS causing a physical change in the molecule. In addition, polymyxin-treated LPS is no longer mitogenic. These results suggest a dissociation between the mitogenic and immunogenic properties of TNP-LPS.