A newly discovered cholesteryl galactoside from Borrelia burgdorferi

Abstract

Two major glycolipids, which comprise Ϸ36% of the total lipid mass from Borrelia burgdorferi, the etiological agent of Lyme disease, were investigated. We determined the fatty acid type, sugar identity, anomeric configuration, and substituent type and position. The structures were identified as cholesteryl 6-O-acyl-␤-D-galactopyranoside (B. burgdorferi glycolipid 1, BbGL-I), and 1,2-di-O-acyl-3-O-␣-D-galactopyranosyl-sn-glycerol (BbGL-II). The major fatty acids were palmitate and oleate. The structures were corroborated by gas-liquid chromatography MS, matrix-assisted laser desorption͞ionization time-of-flight spectroscopy, fast atom bombardment MS, detailed NMR spectrometry, and metabolic labeling. This is a previously undescribed demonstration of a cholesteryl galactoside in bacteria. Lipopolysaccharide was not detected in B. burgdorferi. The two glycolipids have several properties suggesting they may function as lipopolysaccharide: both are main components of the bacterial membrane, surface exposed, and have a three-domain structure. BbGL-I elicited specific antibodies in mice and rabbits, and BbGL-II elicited antibodies that reacted with both glycolipids.T he etiological agent of Lyme disease, Borrelia burgdorferi, is transmitted to humans through the bite of Ixodes ticks. Lyme disease is a multisystem infection, which affects the skin, joints, nervous system, and heart (1). The licensed vaccine (LYMErix, SmithKline Beecham) contains recombinant lipidated OspA. Although lipopolysaccharide (LPS) has been identified in several spirochaetales, such as Leptospira (2) and Treponema (3), there is no evidence for the presence of LPS in Borrelia species (4).Two surface-exposed glycolipids identified in B. burgdorferi react with sera from Lyme disease patients (5-7). These immunoreactive glycolipids have been characterized as monogalactosyl diacylglycerolipids (8). It has been proposed that these glycolipids differ only in their fatty acid composition. We designated these glycolipids as B. burgdorferi glycolipid I (BbGL-I) and B. burgdorferi glycolipid II (BbGL-II). We show that although BbGL-II is a monogalactosyl diacylglycerol as reported (8), BbGL-I has the unique structure cholesteryl 6-O-acyl-␤-D-galactopyranoside. Experimental ProceduresOrganism and Growth Conditions. B. burgdorferi strains B31 (ATCC 35210), BL303 (courtesy of G. Wormser, Division of Infectious Diseases, New York Medical College, Valhalla) and N40 (courtesy of L. Bockenstedt, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT) were cultivated in BSK-H medium (Sigma). Media were inoculated with 2% (vol͞vol) of a frozen culture and incubated statically at 37°C for 72 h to the mid-exponential growth phase. Cells were harvested by centrifugation at 12,000 ϫ g for 30 min, washed three times with cold PBS, and stored at Ϫ20°C.Lipid Extraction and Purification. Lipids were extracted from washed cells as described (9). The chloroform was removed, and the dried lipids (0.1-0.2 mg͞mg cell) dissolved in 1-2 ml of chlorofor...

The bactericidal effect of a complement-independent antibody is osmolytic and specific toBorrelia

“…Because susceptibility to the bactericidal effect is not transferable to other organisms and because there is no obvious accessory protein required for this mechanism, what makes the Borrelia OM so susceptible to the effects of complement-independent bactericidal antibodies? One feature that needs to be considered in the context of OM fluidity in Borrelia is the absence of LPS and the presence of cholesterol-containing glycolipids (27)(28)(29). Because the OM of Borrelia contains cholesterol, a characteristic that is not common for prokaryotes, there is the possibility that lipid raft domains could contribute to increased blebbing and membrane permeability following antibody binding.…”

Hypercholesterolemia and ApoE deficiency result in severe infection with Lyme disease and relapsing-fever Borrelia

“…The borreliae also have cholesterol glycolipids: cholesteryl 6-O-acyl-β-D-galactopyranoside and cholesteryl-β-D-galacto-pyranoside in B. burgdorferi; and 6-O-acylated cholesteryl β-D-glucopyranoside and cholesteryl β-D-glucopyranoside in relapsing-fever Borrelia. They also have noncholesterol glycolipids, monogalactosyl-diacylglycerol in B. burgdorferi and monoglucosyl-diacylglycerol in relapsing-fever Borrelia, as well as many lipoproteins (7)(8)(9)(10)(11)(12)(13)(14)(15). The presence of cholesterol is not common among prokaryotes, but it is increasingly being reported in bacterial pathogens other than Borrelia spp., including species of Helicobacter, Mycoplasma, Ehrlichia, Anaplasma, and Brachyspira (16)(17)(18)(19)(20).…”

Lipid binding orientation within CD1d affects recognition of Borrelia burgorferi antigens by NKT cells

“…These compounds were derived from Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease, and a direct role for iNKT cells in host defense, and clearance of these microbes recently has been demonstrated (15,16). Naturally occurring B. burgdorferi α-galactosyl diacylglycerolipids (α-GalDAG) are composed of two fatty acids, esterified to the sn-1 and sn-2 hydroxyls of glycerol, whereas the galactose is attached to the sn-3 position with an α-glycosidic bond (17). The fatty acids in the purified material, referred to as B. burgdorferi glycolipid-2 (BbGL-2), varied in length from C 14 to C 18 (methylene units) and can either be fully saturated or mono-or di-unsaturated.…”