Natural killer T (NKT) cells recognize glycosphingolipids presented by CD1d molecules and have been linked to defense against microbial infections. Previously defined foreign glycosphingolipids recognized by NKT cells are uniquely found in nonpathogenic sphingomonas bacteria. Here we show that mouse and human NKT cells also recognized glycolipids, specifically a diacylglycerol, from Borrelia burgdorferi, which causes Lyme disease. The B. burgdorferi-derived, glycolipid-induced NKT cell proliferation and cytokine production and the antigenic potency of this glycolipid was dependent on acyl chain length and saturation. These data indicate that NKT cells recognize categories of glycolipids beyond those in sphingomonas and suggest that NKT cell responses driven by T cell receptor-mediated glycolipid recognition may provide protection against diverse pathogens.
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 structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3-dideoxy-␣-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria.The genus Bordetella currently comprises nine species of Gram-negative bacteria. The most extensively studied of these are the respiratory pathogens B. pertussis, B. parapertussis, and B. bronchiseptica. B. pertussis infects only humans and is the causative agent of whooping cough in infants and persistent respiratory infections in adults (1). B. parapertussis exists as two separate lineages. One is adapted to the human host and causes whooping cough; the other is adapted to the ovine host in which it can cause chronic pneumonia (2). In contrast, B. bronchiseptica colonizes the respiratory tract of a large number of animals, and although it causes respiratory infections in some farm, companion, and wild animals, most B. bronchiseptica infections are asymptomatic and chronic. B. bronchiseptica is occasionally isolated from the respiratory tract of humans and is likely acquired through contact with infected animals (3, 4). Although these three pathogens are very closely related genetically (5), they synthesize different lipopolysaccharide (LPS) 2 molecules. All three LPS share similar lipid A and core structures (6), yet only B. parapertussis and B. bronchiseptica synthesize O antigens. Initially, the O antigens of both species were reported to be identical and composed of linear polymers of 1,4-linked 2,3-diacetamido-2,3-dideoxy-␣-galacturonic acid (7), but later differences between the end groups on B. bronchiseptica O antigens were described (8). The core oligosaccharides of B. pertussis and B. bronchiseptica possess an almost identical structure of a branched nonasaccharide with several free amino and carboxyl groups linked to a distal trisaccharide, called band A trisaccharide, whereas the B. parapertussis core comprises a heptasaccharide that lacks band A trisaccharide and two other monosaccharides (9). However, until now, the question of how the O antigens are linked to the core region remained unanswered. Here we present data that describe the complete structures of the B. bronchiseptica and B. parapertussis LPS carbohydrates. EXPERIMEN...
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