The glycolipids of the saprophytic spirochete, Spirochaeta aurantia: A structural, biological, and genetic investigation

Two L&barbelow;arge G&barbelow;lycoL&barbelow;ipids, LGLA and LGLB, were isolated from the saprophytic spirochete, S. aurantia. Initial analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis of LGLA showed a banding pattern reminiscent of smooth lipopolysaccharide (LPS), while LGLB was similar to rough LPS of an S. typhimurium Ra mutant. Hydroxylated fatty acids and 3-deoxy-D-manno-oct-2-ulopyranosonic acid (KDO) were not detected and only LGLB was weakly active in the Limulus amebocyte lysate standard endotoxin assay when compared to control E. coli O111 endotoxin. Neither LGL were able to activate any Toll-like receptor examined. Detailed chemical analysis with nuclear magnetic resonance and mass spectrometry gave the structure of LGLB: R-&parl0;1-4&parr0; -a-GlcA-&parl0;1- 2&parr0;-a-Man-&parl0; 1-4&parr0;-b-GlcA- &parl0;1-4&parr0;-a-G alA6Asp-&parl0;1- a-Fuc3N-&parl0;1- 4&parr0;-b-GlcA- &parl0;1-3&parr0;&flr0; -4&parr0;-a-G alA-&parl0;1-4&parr0;-a -GalNAcA-&parl0;1-4&parr0; -a-GalA-&parl0;1- 3&parr0;-b-Gal-&parl0; 1-1&parr0;-Gro b-Xyl-&parl0; 1-3&parr0;&flr0; b-Xyl- &parl0;1-3&parr0;&flr0; where R = alpha-Fuc3N or alpha-Glc and all monosaccharides were in the D-confirmation. Analysis of the carbohydrate components of LGLA detected large amounts of rhamnose, glucosamine, xylose and fucose that are hypothesized to form the repeating subunit. In addition, a novel sugar that may superficially resemble alpha-2,3 sialic acid was detected in the purpald assay and by reactivity to Ma/II, an alpha-2,3 sialic acid-specific lectin. Analysis of fatty acid composition by gas chromatography and mass spectrometry revealed that the majority were either branched or unsaturated. Lectin blotting demonstrated that S. aurantia also glycosylates the porin and some flagellar subunits.; Freeze substitution electron microscopy showed a structure extending from the surface of S. aurantia cells that could be LGL A. A cluster of open reading frames possessing sequence similarity to proteins known to be involved in the biosynthesis of O-polysaccharide indicate the potential for a Wzy-dependent transport system as well as for the biosynthesis of heptose and 3,6 dideoxyhexose. It is hypothesized that this cluster encodes the biosynthetic apparatus for the LGLA polysaccharide repeat.; This work demonstrates that the outer membrane of S. aurantia does not contain LPS but replaces it with large glycolipids that are structurally similar to LPS but do not contain lipid A. The LGLs are distinct from LPS and likely generate a membrane structure that differs from the classical outer membrane of Gram-negative bacteria.