Characterization of the cell surface glycolipid from Spirochaeta aurantia Catherine J. Paul & Elizabeth A. Lyle & Terry J. Beveridge & Richard I. Tapping & Andrew M. Kropinski & Evgeny Vinogradov Received: 27 September 2008 / Revised: 23 January 2009 / Accepted: 26 January 2009 / Published online: 12 February 2009 # Springer Science + Business Media, LLC 2009 Abstract Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule- like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, L-rhamnose, L-fucose, D-xylose, D-mannose, D-glucosamine, D-glycero- D-gluco-heptose (DDglcHep), D-glycero-D-manno-heptose (DDHep), and a novel branched tetradeoxydecose mono- saccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as:  ½  3  b  DDglcHep  3  b  D  GlcNAc  2  b  D  Man    j a  Aur  3  b  L  Rha  4  b  D  Xyl  4  a  L  Fuc  3  b  DDHep  4 j a  L  Rha  3 which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors. Keywords Glycolipid . Spirochaete . Spirochaeta aurantia . TLR . Structure . NMR Introduction Spirochetes are phylogenetically coherent phylum but show considerable structural, physiological and genomic diversi- ty. With inner and outer membranes and a periplasmic space containing peptidoglycan, spirochetes resemble typ- ical Gram-negative cells though with the unique feature that their flagella are located in the periplasm [1]. One of the major characteristics of the Gram-negative cell is the presence of lipopolysaccharide (LPS, endotoxin), consist- ing of O-antigenic polysaccharide, oligosaccharide core, and a glycolipid “lipid A” [2]. Recognition of LPS by the innate immune system involves at least three receptor molecules: CD14, TLR4 and MD-2. “LPS-sensing appara- tus” also includes heat shock protein, chemokine receptor 4 Glycoconj J (2009) 26:1097–1108 DOI 10.1007/s10719-009-9230-4 C. J. Paul Department of Biotechnology, Lund University, Lund, Sweden, 22100 E. A. Lyle : R. I. Tapping Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA, T. J. Beveridge Department of Molecular & Cellular Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 A. M. Kropinski Department of Microbiology and Immunology, Queen’ s University, Kingston, Ontario, Canada, K7L 3N6 E. Vinogradov (*) Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Dr, Ottawa, Ontario, Canada, K1A 0R6 e-mail: evguenii.vinogradov@nrc.ca