Plant pollen are an important source of antigens that evoke allergic responses. Protein antigens have been the focus of studies aiming to elucidate the mechanisms responsible for allergic reactions to pollen. However, proteins are not the sole active agent present in pollen. It is known that pollen grains contain lipids essential for its reproduction and bioactive lipid mediators. These small molecular compounds are co-delivered with the allergens and hence have the potential to modulate the immune response of subjects by activating their innate immune cells. Previous reports showed that pollen associated lipid mediators exhibited neutrophil- and eosinophil-chemotactic activity and induced polarization of dendritic cells (DCs) toward a Th2-inducing phenotype. In our study we performed chemical analyses of the pollen associated lipids, that are rapidly released upon hydration. As main components we have identified different types of phytoprostanes (PhytoPs), and for the first time phytofurans (PhytoFs), with predominating 16-F_1t-PhytoPs (PPF₁-I), 9-F_1t-PhytoPs (PPF₁-II), 16-E_1t-PhytoPs (PPE₁-I) and 9-D_1t-PhytoPs (PPE₁-II), and 16(RS)-9-epi-ST-Δ¹⁴-10-PhytoFs. Interestingly 16-E_1t-PhytoP and 9-D_1t-PhytoPs were found to be bound to glycerol. Lipid-containing samples (aqueous pollen extract, APE) induced murine mast cell chemotaxis and IL-6 release, and enhanced their IgE-dependent degranulation, demonstrating a role for these lipids in the immediate effector phase of allergic inflammation. Noteworthy, mast cell degranulation seems to be dependent on glycerol-bound, but not free phytoprostanes. On murine dendritic cells, APE selectively induced the upregulation of CD1d, likely preparing lipid-antigen presentation to iNKT cells. Our report contributes to the understanding of the activity of lipid mediators in the immediate effector phase of allergic reactions but identifies a yet undescribed pathway for the recognition of pollen-derived glycolipids by iNKT cells.

Acetobacter pasteurianus is an acetic acid-producing Gram-negative bacterium commonly found associated with plants and plant products and widely used in the production of fermented foods, such as kefir and vinegar. Due to the acid conditions of the bacterium living habitat, uncommon structural features composing its cell envelope are expected. In the present work we have investigated the A. pasteurianus CIP103108 lipopolysaccharide (LPS) structure and immunoactivity. The structure of the lipid A and of two different O-polysaccharides was assessed. Furthermore, immunological studies with human cells showed a low immunostimulant activity of the isolated LPS, in addition to a slight capability to lower the NF-kB activation upon stimulation by toxic LPS.

The Gram-positive lactic acid bacterium Lactobacillus buchneri CD034 is covered by a two-dimensional crystalline, glycoproteinaceous cell surface (S-) layer lattice. While lactobacilli are extensively exploited as cell surface display systems for applied purposes, questions about how they stick their cell wall together are remaining open. This also includes the identification of the S-layer cell wall ligand. In this study, lipoteichoic acid was isolated from the L. buchneri CD034 cell wall as a significant fraction of the bacterium’s cell wall glycopolymers, structurally characterized and analyzed for its potential to mediate binding of the S-layer to the cell wall. Combined component analyses and 1D- and 2D-nuclear magnetic resonance spectroscopy (NMR) revealed the lipoteichoic acid to be composed of on average 31 glycerol-phosphate repeating units partially substituted with α-d-glucose, and with an α-d-Galp(1→2)-α-d-Glcp(1→3)−1,2-diacyl-sn-Gro glycolipid anchor. The specificity of binding between the L. buchneri CD034 S-layer protein and purified lipoteichoic acid as well as their interaction force of about 45 pN were obtained by single-molecule force spectroscopy; this value is in the range of typical ligand–receptor interactions. This study sheds light on a functional implication of Lactobacillus cell wall architecture by showing direct binding between lipoteichoic acid and the S-layer of L. buchneri CD034.

Chemical analyses, mass spectrometry, and NMR spectroscopy were applied to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas veronii strain Bs19, serotype O16. ESI-MS revealed that the most abundant LPS glycoforms have tetra-acylated or hexa-acylated lipid A species, consisting of a bisphosphorylated GlcN disaccharide with an AraN residue as a non-stoichiometric substituent, and a core oligosaccharide composed of Hep₅Hex₃HexN₁Kdo₁P₁. Sugar and methylation analysis together with 1D and 2D ¹H and ¹³C NMR spectroscopy were the main methods used, and revealed that the O-specific polysaccharide (OPS) of A. veronii Bs19 was built up of tetrasaccharide repeating units with the structure: →4)-α-d-Quip3NAc-(1→3)-α-l-Rhap-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→. This composition was confirmed by mass spectrometry. The charge-deconvoluted ESI FT-ICR MS recorded for the LPS preparations identified mass peaks of SR- and R-form LPS species, that differed by Δm = 698.27 u, a value corresponding to the calculated molecular mass of one OPS repeating unit (6dHexNAc6dHexHexHexNAc-H₂O). Moreover, unspecific fragmentation spectra confirmed the sequence of the sugar residues in the OPS and allowed to assume that the elucidated structure also represented the biological repeating unit.

Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease areEscherichia coliandStaphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolateE. coli1303. The structure was determined utilizing chemical analyses, mass spectrometry, and 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[→4)-β-d-Quip3NAc-(1→3)-α-l-Fucp2OAc-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→]- in which theO-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster ofE. coli1303 was also determined. This cluster, located between thegndandgalFgenes, contains 13 putative open reading frames, most of which represent unknown nucleotide sequences that have not been described before. The O-antigen ofE. coli1303 was shown to substitute O-7 of the terminalld-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core – namely terminalld-heptose and glucose were missing; however, it possessed a third Kdo residue in the inner core. On the basis of structural and genetic data we show that the mastitis isolateE. coli1303 represents a new serotype and possesses the K-12 core type, which is rather uncommon among human and bovine isolates.