Accumulation of novel glycolipids and ornithine lipids in Mesorhizobium loti under phosphate deprivation.

Glycolipids are found mainly in photosynthetic organisms (plants, algae, and cyanobacteria), Gram-positive bacteria, and a few other bacterial phyla. They serve as membrane lipids and play a role under phosphate deprivation as surrogates for phospholipids. Mesorhizobium loti accumulates different di- and triglycosyl diacylglycerols, synthesized by the processive glycosyltransferase Pgt-Ml, and two so far unknown glycolipids, which were identified in this study by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy as O-methyl-digalactosyl diacylglycerol (Me-DGD) and glucuronosyl diacylglycerol (GlcAD). Me-DGD is a novel glycolipid, whose synthesis depends on Pgt-Ml activity and the involvement of an unknown methyltransferase, while GlcAD is formed by a novel glycosyltransferase encoded by the open reading frame (ORF) mlr2668, using UDP-glucuronic acid as a sugar donor. Deletion mutants lacking GlcAD are not impaired in growth. Our data suggest that the different glycolipids in Mesorhizobium can mutually replace each other. This may be an adaptation mechanism to enhance the competitiveness in natural environments. A further nonphospholipid in Mesorhizobium was identified as a hydroxylated form of an ornithine lipid with the additional hydroxy group linked to the amide-bound fatty acid, introduced by the hydroxylase OlsD. The presence of this lipid has not been reported for rhizobia yet. The hydroxy group is placed on the C-2 position of the acyl chain as determined by NMR spectroscopy. Furthermore, the isolated ornithine lipids contained up to 80 to 90% d-configured ornithine, a stereoform so far undescribed in bacteria.

NMR-based structural analysis of the complete rough-type lipopolysaccharide isolated from Capnocytophaga canimorsus.

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, (13)C,(15)N labeling could be avoided. The intact LPS was analyzed by homonuclear ((1)H) and heteronuclear ((1)H,(13)C, and (1)H,(31)P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4'-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man(I) residue. This rough-type LPS is exceptional in that all three negative phosphate residues are "masked" by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.

Identification of the flagellin glycosylation system in Burkholderia cenocepacia and the contribution of glycosylated flagellin to evasion of human innate immune responses.

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-D-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.

Unusual outer membrane lipid composition of the gram-negative, lipopolysaccharide-lacking myxobacterium Sorangium cellulosum So ce56.

The gram-negative myxobacterium Sorangium cellulosum So ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. Careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (LPS) are missing in this microbe. Biochemical analysis gave no evidence for the presence of LPS in the membranes of So ce56. By analyzing the lipid composition of its outer membrane sphingolipids were identified as the major lipid class, together with ornithine-containing lipids (OL) and ether lipids. A detailed analysis of these lipids resulted in the identification of more than 50 structural variants within these three classes, which possessed several interesting properties regarding to LPS replacement, mediators in myxobacterial differentiation, as well as potential bioactive properties. The sphingolipids with the basic structure C9-methyl-C(20)-sphingosine possessed as an unusual trait C9-methylation, which is common to fungi but highly uncommon to bacteria. Such sphingolipids have not been found in bacteria before, and they may have a function in myxobacterial development. The OL, also identified in myxobacteria for the first time, contained acyloxyacyl groups, which are also characteristic for LPS and might replace those in certain functions. Finally, the ether lipids may serve as biomarkers in myxobacterial development.

A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity.

Many Gram-positive bacteria produce lipoteichoic acid (LTA) polymers whose physiological roles have remained a matter of debate because of the lack of LTA-deficient mutants. The ypfP gene responsible for biosynthesis of a glycolipid found in LTA was deleted in Staphylococcus aureus SA113, causing 87% reduction of the LTA content. Mass spectrometry and nuclear magnetic resonance spectroscopy revealed that the mutant LTA contained a diacylglycerol anchor instead of the glycolipid, whereas the remaining part was similar to the wild-type polymer except that it was shorter. The LTA mutant strain revealed no major changes in patterns of cell wall proteins or autolytic enzymes compared with the parental strain indicating that LTA may be less important in S. aureus protein attachment than previously thought. However, the autolytic activity of the mutant was strongly reduced demonstrating a role of LTA in controlling autolysin activity. Moreover, the hydrophobicity of the LTA mutant was altered and its ability to form biofilms on plastic was completely abrogated indicating a profound impact of LTA on physicochemical properties of bacterial surfaces. We propose to consider LTA and its biosynthetic enzymes as targets for new antibiofilm strategies.

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands.

UNLABELLED: B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. CONCLUSION: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.

Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.).

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.

Carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens.

Specific IgE binding to carbohydrate moieties of glycosylated allergens has been known for years, but the importance of these structures for the elicitation of allergic reactions is still a matter of debate. Because of their conserved carbohydrate structures, especially N-glycans have always been prime candidates for IgE cross-reactivity between allergens from unrelated species. The aim of our study was to determine whether carbohydrate structures on glycoproteins can by themselves elucidate allergic reactions. We characterized in detail the carbohydrate moieties of the major allergens Phl p 1 and Phl p 13 of timothy grass pollen (Phleum pratense L.) by performing tryptic digests followed by HPLC, N-terminal sequencing, sugar analysis, MALDI-TOF- and ESI-ICRFT-MS. Phl p 1 contains one N-glycan with one of the two glycoforms MMXF3 and M0XF3 and a single furanosidic arabinose, which is bound to a hydroxyproline residue in direct vicinity to the N-glycan. This O-glycosylation is probably due to an arabinosylation consensus sequence found in the N-terminal part of Phl p 1 and other group 1 allergens, but displayed no IgE-reactivity. Thus, Phl p 1 is monovalent with respect to its IgE-binding carbohydrate epitopes and showed no mediator release. In contrast, the carbohydrate moiety of Phl p 13, which carries four of the same N-glycans (like Phl p 1), can cross-link IgE-receptors via carbohydrate chains and elicits IL-4 release from basophils.

The obligate predatory Bdellovibrio bacteriovorus possesses a neutral lipid A containing alpha-D-Mannoses that replace phosphate residues: similarities and differences between the lipid As and the lipopolysaccharides of the wild type strain B. bacteriovorus HD100 and its host-independent derivative HI100.

Bdellovibrio bacteriovorus are predatory bacteria that penetrate Gram-negative bacteria and grow intraperiplasmically at the expense of the prey. It was suggested that B. bacteriovorus partially degrade and reutilize lipopolysaccharide (LPS) of the host, thus synthesizing an outer membrane containing structural elements of the prey. According to this hypothesis a host-independent mutant should possess a chemically different LPS. Therefore, the lipopolysaccharides of B. bacteriovorus HD100 and its host-independent derivative B. bacteriovorus HI100 were isolated and characterized by SDS-polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometry. LPS of both strains were identified as smooth-form LPS with different repeating units. The lipid As were isolated after mild acid hydrolysis and their structures were determined by chemical analysis, by mass spectrometric methods, and by NMR spectroscopy. Both lipid As were characterized by an unusual chemical structure, consisting of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide carrying six fatty acids that were all hydroxylated. Instead of phosphate groups substituting position O-1 of the reducing and O-4' of the nonreducing end alpha-d-mannopyranose residues were found in these lipid As. Thus, they represent the first lipid As completely missing negatively charged groups. A reduced endotoxic activity as determined by cytokine induction from human macrophages was shown for this novel structure. Only minor differences with respect to fatty acids were detected between the lipid As of the host-dependent wild type strain HD100 and for its host-independent derivative HI100. From the results of the detailed analysis it can be concluded that the wild type strain HD100 synthesizes an innate LPS.