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In this work, we carried out the elucidation of a novel lipooligosaccharide LOS from a clinical strain of the most virulent strain of the Burkholderia cepaciacomplex, which was of B. cenocepaciaET12 epidemic strain lineage. The strain was isolated from a cystic fibrosis patient who had successfully undergone lung transplantation. This molecule presents some specific chemical differences when compared to the LOS reported so far from other B. cenocepaciaET12 clones. The new endotoxin was also tested as a cytokine enhancer on human myelomonocytic U937 cells. Lipopolysaccharides are the main virulence factors of Gramnegative bacteria, and since they are exposed to the extracellular space, they are deeply involved in bacterial adaptation to the host environment. The characterisation of structural motifs present in these molecules is essential to the comprehension of the persistencesurvival mechanisms and inflammatory potential of these bacteria.

The complete structure of the lipopolysaccharide isolated from the Gram-negative marine bacterium Alteromonas addita, type strain KMM 3600T = R10SW13T, has been elucidated by means of a combined chemical approach and state-of-the-art NMR and MS analyses. Isolation and characterisation of the lipid A moiety and the core oligosaccharide were pursued separately after either acid or alkaline treatment of the lipopolysaccharide. The structure detected was identified as a novel, highly negatively charged, deep-rough lipopolysaccharide in which a trisaccharide subunit is connected to a typical lipid A glucosamine backbone. Within the core oligosaccharide, a phosphodiester bridge connects a glucose unit to a heptose residue.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

This paper deals with the full structural elucidation of the lipooligosaccharide from the Gram-negative bacterium Burkholderia pyrrocinia. B. pyrrocinia is one of the nine species included in the Burkholderia cepacia complex (BCC), a group of important opportunistic pathogens in patients with cystic fibrosis and chronic granulomatous disease. B. pyrrocinia strain BTS7, isolated from a patient with cystic fibrosis, was found to exclusively produce a lipooligosaccharide (LOS). This component of the external membrane plays a key role in the virulence of BCC bacteria and is required for resistance to antimicrobial compounds and for bacterial survival. Here we present, for the first time, a detailed study of the structure of the lipooligosaccharide (LOS) elucidated by means of compositional analysis, MALDI and ESI mass spectrometry and 2D NMR spectroscopy. The LOS of B. pyrrocinia was degraded by complete deacylation, dephosphorylation and reduction. The major oligosaccharide representing the carbohydrate backbone was isolated by size-exclusion chromatography and identified. The structural determination of molecules (such as the LOS of B. pyrrocinia) involved in the activation of the pro-inflammatory processes is the first step in the adoption of new therapeutic strategies against lung infections.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Alteromonas macleodii ATCC 27126T is a Gram-negative marine bacterium isolated from a sea water sample collected from around the Hawaiian Islands. The structure of the lipooligosaccharide derived from its outer membrane has been fully determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-O-acylation) followed by de-N-acylation using hot KOH and furnished a single core glycoform. Mild acid hydrolysis was employed to obtain the lipid A moiety which was selectively de-O-acylated and analysed to determine its primary structure. The structural elucidation of both fractions was carried out by chemical analyses, 2D NMR spectroscopy and MALDI-TOF mass spectrometry and revealed a novel lipooligosaccharide with an unusual structure. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Shewanella pacifica is a Gram-negative microrganism that is able to grow in sea water. A novel lipooligosaccharide (LOS) has been isolated from the outer membrane of this bacterium and its primary structure fully characterised. For the first time, the presence of a 2,3-dihydroxypropanoic acid residue (glyceric acid) has been identified in the core region. The complete structure of the LOS was determined by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H, 13C and 31P NMR spectroscopy on the oligosaccharides formed by selective degradation of the LOS. Strong alkaline treatment, aimed at recovering and identifying the complete carbohydrate backbone, was carried out by hydrazinolysis followed by de-N-acylation with hot KOH, whereas mild hydrazinolysis (de-O-acylation) allowed us to gain information about the nature of the phosphate and other non-carbohydrate substituents on the core oligosaccharide. Mild acid hydrolysis was employed to obtain a lipid A moiety with which further degradation and mass spectrometry experiments were carried out in order to determine its primary structure. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Two O-chain structures were identified after acid hydrolysis of the lipopolysaccharide fraction of Agrobacterium radiobacter (type strain). The first is constituted by the linear tetrasaccharide repeating unit [2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→]n and the second by the (1→2)-branched repeating unit α-D-Manp-(1→2)−[3)-α-D-Fucp-(1→3)-α-D-Fucp-(1→]n.The two structures were determined mainly by 1D- and 2D NMR spectroscopy together with chemical-degradation methods. A detailed analysis of the NOESY spectrum, supported by molecular dynamics calculations, suggested that some unexpected NOEs were due to the sum of many small dipolar effects, whose identification was possible only by considering the 3D structure of an O-chain oligosaccharide bigger than the repeating unit. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)