doi:10.1111/j.1432-1033.2004.04433.x
Eur. J. Biochem. 271, 4685–4695 (2004) (cid:1) FEBS 2004
The structure and biological characteristics of the Spirochaetaaurantiaouter membrane glycolipid LGLB
Evgeny Vinogradov1, Catherine J. Paul2, Jianjun Li1, Yuchen Zhou2, Elizabeth A. Lyle3, Richard I. Tapping3, Andrew M. Kropinski2 and Malcolm B. Perry1 1Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada; 2Queen’s University, Kingston, ON, Canada; 3University of Illinois, Urbana, IL, USA
contained two fatty groups at O-2 and O-3 of the glycerol residue. Nonhydroxylated C14 to C18 fatty acids were identified, which were predominantly unsaturated or bran- ched. LGLB was able to gel Limulus amebocyte lysate, albeit at a lower level than that observed for Escherichia coli O113 lipopolysaccharide. However, even large amounts of LGLB were unable to stimulate any Toll-like receptor (TLR) examined, including TLR4 and TLR2, previously shown to be sensitive to lipopolysaccharide and glycolipids from diverse bacterial origins, including other spirochetes.
Keywords: glycolipid; Spirochaeta aurantia; structure.
In an attempt to isolate lipopolysaccharide from Spirocha- eta aurantia, Darveau-Hancock extraction of the cell mass was performed. While no lipopolysaccharide was found, two carbohydrate-containing compounds were detected. They were resolved by size-exclusion chromatography into high molecular mass (LGLA) and low molecular mass (LGLB) fractions. Here we present the results of the analysis of the glycolipid LGLB. Deacylation of LGLB with hydrazine and separation of the products by using anion-exchange chro- matography gave two major products. Their structure was determined by using chemical methods, NMR and mass spectrometry. All monosaccharides had the D-configuration, and aspartic acid had the L-configuration. Intact LGLB
T. medium [11], and T. denticola [12]) possess a surface glycolipid similar to the lipotechoic acid of Gram-positive bacteria. Recently, several small surface glycolipids were identified in B. burgdorferi [13,14].
Toll-like receptors (TLR) are an important component of the host response to invading bacteria, with TLR4 required for signal transduction and the inflammatory response following exposure of cells to LPS derived from Gram- negative enteric bacteria [15–17]. Although LPS derived from enteric bacteria is a potent agonist for TLR4, other nonenteric bacterial LPS, such as that derived from Legionella pneumophila, Leptospira interrogans and at least one strain of Porphyromonas gingivalis can act as agonists for TLR2 [8,18,19].
Spirochetes are a group of bacteria unified by spiral or flattened-waveform cell morphology and periplasmic endo- flagella; Spirochaeta is one of the six genera within this phylum [1]. This bacterium is a free-living nonpathogenic spirochete, originally isolated from pond mud and able to fix atmospheric nitrogen [2–4]. Other members of this phylum include the human pathogens Borrelia burgdorferi (Lyme disease), the Leptospira (leptospiroses), Treponema pallidum (syphilis), and T. denticola, T. brennaborense, and T. maltophilum, which are implicated in periodontal disease [5–7]. Although classified as Gram-negative, controversy exists over the existence of lipopolysaccharide (LPS) in the outer membranes of spirochetes. Clear genetic and bio- chemical evidence exists for the presence of LPS in Leptospira [8] and for its absence in T. pallidum and Borrelia [9,10]. Limited structural analysis suggests that several oral treponemes (T. brennaborense and T. maltophilium [6],
The glycolipids isolated from T. denticola, T. brennabo- rense, and T. maltophilum appear to have functional similarity to LPS in that they possess some ability to gel Limulus amebocyte lysate (LAL) [12,20], a standard assay for endotoxin activity. In addition, while glycolipid derived from T. brennaborense stimulates immune cells through TLR4, the glycolipids from T. denticola and T. maltophilum stimulate cells through TLR2 [5,6,20]. The strict correlation between the structure of the LPS molecule with that of TLR specificity remains undefined but it is clear that TLR2 is capable of recognizing a wider range of potential lipid A structures than TLR4 [21].
S. aurantia has simple growth requirements that facilitate studies otherwise limited by the amount of cell mass, a problem often limiting studies on other spirochetes [2]. We describe here the structural characterization of the carbo- hydrate skeleton and fatty acids of one of its glycolipids, LGLB. In addition we present evidence which suggests that
Correspondence to E. Vinogradov, Institute for Biological Sciences, National Research Council, 100 Sussex Dr., Ottawa, ON, Canada K1A 0R6. Fax: +1 613 952 9092, Tel.: +1 613 990 0832, E-mail: evguenii.vinogradov@nrc-cnrc.gc.ca Abbreviations: EU, endotoxin units; FAME, fatty acid methyl esters; GalNAcA, N-acetylgalactosaminuronic acid; GSL, glycosphinogo- lipids; Fuc3N, 3-amino-3,6-dideoxygalactose; Kdo, 2-keto-3-deoxy- D-manno-oct-2-ulosonic acid; LAL, Limulus amebocyte lysate; LBP, LPS-binding protein; LPS, lipopolysaccharide; SGM, spirochaete growth medium; TLR, Toll-like receptor; TNF-a, tumour necrosis factor-a. (Received 9 August 2004, revised 30 September 2004, accepted 13 October 2004)
4686 E. Vinogradov et al. (Eur. J. Biochem. 271)
(cid:1) FEBS 2004
NMR spectroscopy and general methods
while superficially resembling other spirochetal glycolipids, LGLB is a multisaccharide glycolipid and is unable to stimulate any TLR examined.
Experimental procedures
Bacterial strain and growth conditions
NMR spectra were recorded at 25 (cid:2)C in D2O on a Varian UNITY INOVA 600 instrument using acetone as the external reference (1H, 2.225 p.p.m., 13C, 31.45 p.p.m.). Varian standard programs COSY, NOESY (mixing time of 300 ms), TOCSY (spinlock time 120 ms), HSQC, HMQCTOCSY, and gHMBC (evolution delay of 100 ms), were used.
The S. aurantia strain, M1, used in this study, was obtained originally from E. P. Greenberg (Ohio State University, Columbus, OH, USA). It was propagated in spirochete growth medium (SGM) containing 0.4% (w/v) maltose (Sigma-Aldrich, St. Louis, MO, USA), 0.2% (w/v) tryptone and 0.2% (w/v) yeast extract (Difco), at pH 7.5. Cells were grown at 30 (cid:2)C with gentle aeration (30 r.p.m.; orbital shaker; Forma Scientific, Marietta, OH, USA) for 24–48 h. Cell stocks were maintained in SGM in liquid nitrogen.
Glycolipid isolation
Capillary electrophoresis-electrospray mass spectrometry (CE-MS). Mass spectrometric experiments were conduc- ted by using a Q-Star Quadropole/time-of-flight instrument (Applied Biosystems/Sciex, Concord, ON, Canada). Briefly, samples were analyzed on a crystal Model 310 CE instrument (ATI Unicam, Boston, MA, USA) coupled to a Q-Star via a microIonspray interface. A sheath solution (isopropanol/methanol, 2 : 1, v/v) was delivered at a flow rate of 1 lLÆmin)1 to a low dead volume tee. The separation was obtained on a bare fused-silica capillary, of (cid:1) 90 cm length, using 10 mM ammonium acetate/ammonium hydroxide in deionized water, pH 9.0, containing 5% (v/v) methanol. A voltage of 25 kV was typically applied at the injection. Mass spectra were acquired with dwell times of 2.0 s per scan in positive ion detection mode. Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell, were recorded by a time-of-flight mass analyzer. Collision energies were typically 120 eV (laboratory frame of refer- ence).
Isolation of LGL from S. aurantia. Bacteria were harves- ted from a total of 55 L of SGM and the combined cell pellet was extracted following the method of Darveau & Hancock [22]. The final product was extracted once with cold 95% (v/v) ethanol and twice with chloroform/meth- anol (2 : 1, v/v) to remove phospholipids and carotenoids. The residue was resuspended in distilled water, and contaminating protein was removed by treatment with pronase (25 lgÆmL)1) for 18 h at 37 (cid:2)C. A final extraction by chloroform/methanol (2 : 1, v/v) was followed by dialysis against distilled water using Slide-a-lyzer(cid:3) 10K cassettes (Pierce Chemical Company, Rockford, IL, USA) and lyophilization. The overall yield was determined by comparing the mass of a white powdery material left after dialysis and lyophilization (547 mg), to the original dry weight of lyophilized whole cells of S. aurantia from which that glycolipid material had been isolated (3.51 g).
Hydrolysis. Hydrolysis was performed with 4 M CF3CO2H (110 (cid:2)C, 3 h), monosaccharides were conventionally con- verted into the alditol acetates and analysed by GLC on an Agilent 6850 chromatograph equipped with a DB-17 (30 m · 0.25 mm) fused-silica column using a temperature gradient of 180 (cid:2)C (2 min) fi 240 (cid:2)C, at 2 (cid:2)CÆmin)1. GC- MS was performed on the Varian Saturn 2000 system with an ion-trap mass spectral detector using the same column.
(1.6 · 80 cm)
Gel chromatography. Gel chromatography was carried out on Sephadex G-50 (2.5 · 95 cm) and Sephadex G-15 in pyridinium-acetate buffer, columns pH 4.5 (4 mL of pyridine and 10 mL of AcOH in 1 L of water), and the eluate was monitored by a refractive index detector.
Configuration experiments
Column chromatography. Crude LGL (1.5 g) was dis- solved in sample buffer [20 mM Tris/HCl, pH 8; 50 mM EDTA; 10% w/v) SDS] and fractionated on a 5.5 · 40 cm column of Sephacryl S-300 HR (Sigma-Aldrich) at 25 (cid:2)C [column buffer: 10 mM Tris/HCl, pH 8; 10 mM EDTA; 0.2 M NaCl; 0.3% (w/v) SDS]. Fractions of (cid:1) 2.1 mL were collected at an average flow rate of 1.5 mLÆmin)1. The fractions containing the low molecular mass material (LGLB), as determined by standard SDS/PAGE with silver stain [23], were pooled, precipitated with cold 0.375 M MgCl2 in 95% (w/v) ethanol, suspended in distilled water and subjected to a second chromatography to ensure homogeneity. Material was then reprecipitated, suspended lyophilized and weighed in in distilled water, dialyzed, preparation for further analysis.
For determining the absolute configuration of the mono- saccharides, product 2 (1 mg) was treated with (S)-2- butanol/AcCl (0.25 mL, 10 : 1, v/v) for 2 h at 85 (cid:2)C, dried under the stream of air, acetylated and then analysed by GC in comparison with authentic standards, prepared from the respective monosaccharides with (S)- and (R)-2-butanol.
Tricine–SDS/PAGE. Tricine–SDS/PAGE [15% (w/v) resolving gel; 10% (w/v) spacer gel; 4.5% (w/v) stacking gel) was used to examine the low molecular mass portions of LPS and LGL [24]. LPS from Salmonella enterica sv. typhimurium wild type, Sal. enterica sv. typhimurium TV 119 (Ra mutant) and Sal. enterica sv. minnesota R5 (Rc mutant) were purchased from Sigma-Aldrich. Products in acryl- amide gels were visualized by silver staining [23].
For determination of the configuration of N-acetylgalac- tosaminuronic acid (GalNAcA), a sample (2 mg) of LGLB was treated with 1 M HCl in methanol (100 (cid:2)C, 4 h), dried, and then the product was peracetylated by Ac2O in pyridine (0.5 + 0.5 mL, at 85 (cid:2)C for 30 min) and reduced with excess NaBD4 in 96% (v/v) ethanol (1 mL) at 40 (cid:2)C. Acetic acid (1 mL) was added, the product was dried under a
(cid:1) FEBS 2004
Analysis of Spirochaeta aurantia glycolipid LGLB (Eur. J. Biochem. 271) 4687
stream of air and then dried twice with the addition of 1 mL of methanol to remove boric acid. (R)-2-BuOH (0.5 mL) and AcCl (0.08 mL) were added to the dry residue, the mixture was incubated for 4 h at 85 (cid:2)C, filtered, dried, acetylated by Ac2O in pyridine (0.5 + 0.5 mL, at 85 (cid:2)C for 30 min), dried and analyzed by GC-MS with the standards prepared from D-GalN and (R)- and (S)-2-BuOH.
The absolute configuration of L-aspartic acid was deter- mined by chiral HPLC of the oligosaccharide hydrolysate on a Chirex D penicillamine column (250 · 4.6 mm; Phenomenex) in 15% (v/v) methanol containing 2 mM CuSO4, with UV detection at 254 nm.
standards. Agonists
examined were
carried out at 37 (cid:2)C in an atmosphere of 5% carbon dioxide for 6 h. Cell supernatants were removed and assayed for cytokine production by standard sandwich ELISA in 96-well Immunlon plates (Dynatech Laboratories, Chant- illy, VA, USA). The TNF-a ELISA was performed by using mAbs 68B6A3 or 68B2B3 for capture and the biotinylated mAb 68B3C5 (Biosource International, Camarillo, CA, USA), followed by streptavidin-conjugated horseradish peroxidase (HRP), for detection. ELISAs were developed by using o-phenylenediamine as a substrate, and the absorbance was measured at 490 nm by using a Spectramax plate reader and software (Molecular Devices, Sunnyvale, CA, USA). All values were interpolated from either a log- log or a four-parameter fit of a curve generated from the appropriate S. aurantia LGLB (5 lgÆmL)1), zymosan (5 · 109 particles per mL; Molecular Probes, Eugene, OR, USA), heat-killed Staphylococcus aureus (2.5 · 106 particles per mL; Molecu- lar Probes), PolyIC (50 lgÆmL)1; Sigma Genosys, The Woodlands, TX, USA), E. coli Re595 LPS (20 ngÆmL)1; repurified as decribed previously [26]), R848 (1 lgÆmL)1; Invivogen, San Diego, CA, USA) and CpG Oligo (2 lM; Sigma Genosys).
Results
Fatty acid methyl esters (FAMEs) were generated from 1 mg samples of LGLB by the addition of 1 mL of 3 M HCl in methanol (Alltech Associates, Inc., Deerfield, IL, USA) and incubation at 100 (cid:2)C for 18 h. Following liberation of the FAMEs, the hydrolysates were neutralized with 0.46 g of silver carbonate and doped with 204.5 lg of tridecanoic acid (in n-pentanol) as an internal standard. The samples were centrifuged and the FAMEs were resolved by PerkinElmer Sigma 3 gas chromatography, equipped with a glass column [3.05 m · 2 mm internal diameter, packed with 3% (w/v) SP-2100 DOH, 100/120 Supelcoport with carrier gas (N2)], at a flow rate of 50 mlÆmin)1. The oven was programmed as follows: 150 (cid:2)C for 5 min; followed by 150(cid:2) to 230(cid:2) C at 8(cid:2) CÆmin)1. Data analysis was conducted by using the PEAKFIT(cid:3) v. 4.11 software package (Systat Software Inc., Richmond, CA, USA). Comparison of FAME retention times with those of a Bacterial Acid Methyl Esters CPTM mix (Matreya, Inc., Pleasant Gap, PA, USA) permitted tentative FAME identifications to be made. The latter were confirmed by GLC-MS analysis (Analytical Services, Queen’s University, Kingston, ON, Canada).
LGLB was O-deacylated by hydrazinolysis, as described by Gu et al. [25]. Briefly, 40 mg of LGLB was incubated with anhydrous hydrazine for 3 h at 37 (cid:2)C, with occasional mixing. The mixture was then chilled to )20 (cid:2)C and an equal volume of chilled acetone was added dropwise. The product was recovered by centrifugation, washed once with chilled acetone, dried and weighed.
Darveau-Hancock extraction of stationary phase S. auran- tia cells gave a white powdery substance in a yield of 15.6% based upon the cell dry wieght. This high yield is not unexpected as the surface to volume ratio of this bacterium is 13.6Ælm)1, approximately 3.5 times higher than that of E. coli or Sal. enterica sv. typhimurium (3.9Ælm)1). The Darveau-Hancock procedure does not discriminate between high ((cid:1)smooth(cid:2)) or low ((cid:1)rough(cid:2)) molecular mass LPS, provides a high yield of product and should apply equally to polysaccharides or glycolipids [22]. Potential complex glycolipids were separated from previously characterized glycogen storage granules by size exclusion chromatography with examination of the fractions for carbohydrates and hexosamines [27]. A low molecular mass carbohydrate- containing material (LGLB) was isolated, and when examined by Tricine–SDS/PAGE [24], demonstrated mobil- ity similar to the rough LPS of a Sal. enterica sv. typhimurium TV 119 Ra mutant (Fig. 1). Another material, LGLA, was identified as a larger glycolipid and is thought to contain O-antigen like repeats, contributing to the banding pattern observed in crude S. aurantia extract (data not shown).
Preliminary colorimetric analysis [28]
Separation of oligosaccharides 1 and 2 was performed by ion-exchange chromatography on a Hitrap Q anion- exchange column containing 5 mL of Q-Sepharose Fast Flow (Amersham Pharmacia Biotech) in a gradient of water/1 M NaCl over 1 h with UV detection at 220 nm. The products were desalted by gel chromatography on a Sephadex G-15 column.
Biological assays
indicated that LGLB did not contain any 2-keto-3-deoxy-D-manno-oct-2- ulosonic acid (Kdo). The material was subjected to methanolysis [29], and the fatty acid methyl esters were analyzed by GLC, revealing five major acyl constituents, none of which were the characteristic hydroxylated fatty acids of LPS (Table 1).
LGLB was O-deacylated by treatment with anhydrous hydrazine, and the oligosaccharides were separated by anion-exchange chromatography to give two main compo- nents (1 and 2), which differed by one monosaccharide residue. Their structure was determined by NMR spectros- copy, MS and chemical analysis. Monosaccharide analysis (GC of alditol acetates or acetylated products of acidic methanolysis) of both products showed that their compo-
LAL assays were conducted by the Associates of Cape Cod, Inc. (Cape Cod, MA, USA), by using the gel-clot method, and the number of endotoxin units (EU) was compared with control standard endotoxin from Escherichia coli O113. The activation of TLRs was measured by quantifying the production of tumour necrosis factor-a (TNF-a) by whole blood cells, in response to a panel of TLR agonists, as described by Tapping et al. [26]. Briefly, whole blood from healthy donors was collected into tubes containing heparin and diluted 1 : 4 in RPMI 1640. Samples were aliquoted into 96-well plates, agonist was added, and incubation was
4688 E. Vinogradov et al. (Eur. J. Biochem. 271)
(cid:1) FEBS 2004
tified glucuronic, galactosaminouronic, and galacturonic acids. The presence of excess glucose (glucitol) in alditol acetate analysis is a result of the reduction of glucurono- lactone. The 3-amino-3,6-dideoxyhexose is quantified approximately because of the lack of a quantitative standard compound.
1
5
2
3
4
Fig. 1. Visualization of LGLB by Tricine–SDS/PAGE and silver staining reveals that this material co-electrophoreses with the Ra form of lipopolysaccharide (LPS) from Salmonella enterica sv. typhimurium. Lane 1, wild-type Sal. enterica sv. typhimurium LPS, 30 lg; lane 2, Spirochaeta aurantia crude LGL, 20 lg; lane 3, S. aurantia LGLB, 15 lg; lane 4, Sal. enterica sv. typhimurium TV119 (Ra mutant), 2 lg; lane 5, Sal. enterica sv. minnesota R5 (Rc mutant), 2 lg.
Table 1. Fatty acid methyl ester (FAME) analysis, GLC and GLC-MS indicated that the majority of fatty acids contained in Spirochaeta aurantia LGLB are either branched or unsaturated. Values stated are the average nmolÆmg)1 with standard deviations (±) obtained from quantifying and averaging areas under specific peaks from GLC analysis of four separate samples of LGLB.
NMR spectra of both oligosaccharides were completely assigned by using 2D NMR techniques (Figs 2–4, Table 2). Monosaccharides were identified on the basis of vicinal proton coupling constants and 13C NMR chemical shifts. Anomeric configurations were deduced from the J1,2 coupling constants and chemical shifts of H-1, C-1 and C-5 signals. The position of C-6 signals of uronic acids was found from HMBC correlations to H-5 protons. Connec- tions between monosaccharides were identified on the basis of NOESY (Fig. 3) and HMBC correlations. The following inter-residual NOEs were observed in oligosaccharides 1 and 2: P1G4 (in 1), C1G4 (in 2), and G1A2, G1A1, A1G5, A1L4, A1L3, L1E4, E1F4, F1I4, I1D4, D1N3, D1N4, N1Q1, B1K4, K1E3, O1I3, and M1D3. These correlations include several contacts to nontransglycosidic protons next to the linkage position, and between H-1 of a monosac- charide and H-5 of a glycosylating residue in the event of an a-(1–2)-linkage. Respective HMBC correlations between H-1 and a carbon at the transglycosidic position were identified for all linkages. Amide linkage between C-6 of residue E and an amino group of the aspartic acid was identified on the basis of the HMBC correlation between H-2 of aspartic acid and C-6 of the GalA E, thus showing that aspartic acid is amide linked through its amino group to C-6 of galacturonic acid E (Fig. 4).
Identity of fatty acid LGLB (nmolÆmg)1)
sition was similar, comprising glycerol, xylose, mannose, glucose, galactose, and 3-amino-3,6-dideoxyhexose in a ratio of 1 : 2.5 : 1 : 1.6 : 1 : 1.5, and additionally nonquan-
Absolute configuration of the monosaccharides was determined by GC analysis of acetylated 2-butyl glycosides. For 3-amino-3,6-dideoxygalactose (Fuc3N), the O-specific polysaccharide from Proteus penneri 16 was used as a source of reference D-Fuc3N, where its D-configuration was determined earlier [30]. For determining the configuration of GalNAcA, an LGLB sample was treated with HCl in
Tetradecanoic acid (C14:0) 13-Methyltetradecanoic acid (iC15:0) 15-Methylpentadecanoic acid (iC16:0) 9-Hexadecenoic acid (C16:19) 9-Octadecenoic acid (C18:19) 34.9 ± 1.3 224.1 ± 9.0 117.6 ± 12.4 155.3 ± 10.5 58.3 ± 5.6
2
1
5
4
3
2
[ppm]
Fig. 2. 1H NMR spectra of oligosaccharides 1 and 2.
(cid:1) FEBS 2004
Analysis of Spirochaeta aurantia glycolipid LGLB (Eur. J. Biochem. 271) 4689
ppm
TOCSY
NOESY
M15 O15
O12 O15
L12
O13
O13
M15 M12 M13
C14
K12
K12
C12
C12
G12
O14
B13
G12
A1:L3
M14
3.7
L13,15
C13
C1:G4
N12
N15
G14
N1:Q1
L14
F24
F12
C15
A1:L4
E5:F2
F12
B1:K4
N13
N13
K13 K14,15
K15
D1:N3
M15
B12
O15
B12,14
G13
G1:A2
I34
O1:I3
I13
I34
E24
E12
E12
A12
I23
A12
E13
E35
E34
4.2
D12
D12,13
K1:E3
F34
F34
D34
F13 G15
I35
N14
D1:N4
A1:G5
M1:D3
I12
I24
I12
E1:F4
F45
F45
F14
I1:D4
I45
I14
D45
D15 D14
4.7
F1:I4
E45
E14
E45
E15
A1:G1
5.2
5.6
5.4
5.2
5.0
4.8
4.6 ppm
5.6
5.4
5.2
5.0
4.8
4.6 ppm
methanol, and the product was peracetylated in order to reacetylate free amino groups; it was checked by GC-MS for the presence of methyl ester of methyl GalNAc. The product was reduced with NaBD4 in 96% (v/v) ethanol at 40 (cid:2)C, treated with HCl-(R)-2-BuOH, acetylated and ana- lysed by GC-MS. A total ion chromatogram showed no well pronounced peaks; however, a fragmentogram for the expected glycosyl cation of m/z 332 contained peaks with the same retention time as that obtained from D-GalN with (R)-2-BuOH; mass spectra of the products obtained from LGLB were shifted to high mass by two units owing to a double deuteration at C-6. Thus, GalNA had the D-configuration.
These data, taken together, allow us to propose the structures of oligosaccharides 1 and 2, as presented in Scheme 1.
pound 2 with m/z 1197.25 was observed as the most abundant ions (observed molecular mass: 2392.50 Da). The composition details, as well as some sequence information of those two major components with m/z 1180.24 and 1197.25, were further characterized by tandem mass spectrometry (MS/MS). The fragmentation of cationic oligosaccharides typically proceeds by cleavage at the glycosidic bonds, which provides sequence and branching information [31]. The charge state of a fragment ion is then identified by using the isotope profile, owing to the high resolution provided by the TOF mass analyser. The product-ion spectrum (MS/MS spectrum), obtained from a doubly charged ion at m/z 1197.25, is illustrated in Fig. 6. This spectrum revealed two major doubly charged ions at m/z 503.63 and 672.67, which corresponds to the single charged ion at m/z 1006.27 and 1344.37, respectively. In addition, a series of single charged ions was generated via the formation of complementary fragment pair of B and Y ions. As indicated in Fig. 6, the fragment ion at m/z 1876.56 corresponded to the loss of the nonreducing end C-G-A unit and ammonium from the molecular ion. Further fragmentation gave the fragment ion at m/z 1436.42, owing to the loss of the branching xylose residues M and O, and of GlcA residue L. The remaining linear sequence, consisting of K-B-F-I-D-N-Q, was confirmed by the observation of
The oligosaccharides were further analyzed by CE-MS and by CE-MS/MS (Figs 5 and 6). The mass spectra obtained in positive ion detection mode for oligosaccharide 1 showed a major doubly charged ion at m/z 1180.25 (observed molecular mass: 2358.50 Da; calculated exact molecular mass for C85H130O72N4: 2358.6633 Da). The MS for oligosaccharide 2 showed a molecular mass of 2375.56 Da (calculated exact mass for C85H129O74N3: 2375.6422). In addition, an ammonium adduct of com-
Fig. 3. Fragments of TOCSY (left) and NOESY (right) spectra of oligosaccharide 2. Intraresidual correlations are labeled with a letter designation of the monosaccharide residue and numbers of the correlating protons. Inter-residual correlations are labeled with letters for both monosaccharides.
4690 E. Vinogradov et al. (Eur. J. Biochem. 271)
(cid:1) FEBS 2004
Asp3
HSQC
40
Asp2
60
E5
80
100
Asp12;Asp24
E56
Asp2:E6
HMBC
170 174 178
5.5
5.0
4.5
4.0
Asp13;Asp34 3.0
3.5
Fig. 4. Fragments of HSQC and HMBC spectra of compound 1. Labels illustrate assignment of the amide linkage between the amino group of Asp and the carboxyl group of GalA residue E.
detergent. These results show that no additional acylation, except at the glycerol residue, is present in the oligosac- charides.
LGLB does not activate any Toll-like receptor
fragment ions at m/z 292.06, 468.09, 613.16, 789.21, 1006.27, 1182.24, and 1344.37, respectively. The fragment ion at m/z 556.15 corresponds to the unit I-D-N, which might result from the loss of Q from I-D-N-Q (m/z 648.20) or from the loss of F from F-I-D-N (m/z 732.18). However, many other combinations of fragments are also possible, because of the existence of branches in the molecule. Similarly, the tandem MS was conducted for the doubly charged ion at m/z 1180.25 (data not shown) and the mass spectral data fully agree with the sequence determined by NMR.
The gelation of LAL is a standard assay based on the nonspecific immune response of the horseshoe crab, and is used to assess the endotoxic potential of various substances [32]. LGLB displayed a 100-fold less endotoxic potential, registering 2.5 · 105 EUÆmg)1 when compared to an E. coli O113 LPS control (1 · 107 EUÆmg)1) in a LAL gel clot assay.
(zymosan and heat-killed Staph. aureus
Knowledge of the deacylated oligosaccharide structures allowed analysis of intact glycolipids by NMR. Spectra of reasonable quality were obtained at 60 (cid:2)C in the presence of 5% fully deuterated SDS. All monosaccharides present in the products 1 and 2 were identified, and the ratio of structures 1 and 2 was close to 1 : 1. All chemical shifts remained mostly unchanged with the exception of H/C-2 and H/C-3 of the glycerol residue. Proton signals were strongly downfield shifted owing to acylation (Table 2); 13C signals also experienced downfield substitution effects. No data regarding attachment of particular acyl groups at O-2 and O-3 of the glycerol residue was obtained. Several attempts to obtain a mass spectrum of the LGLB by using CE-MS, ESI-MS and MALDI were unsuccessful, prob- ably because this compound is not soluble without a
LGLB was also examined for its ability to act as a TLR agonist. Attempts to measure a reaction from cells trans- fected specifically with human TLR2 or TLR4 were unsuccessful, regardless of the concentration of LGLB examined (data not shown). The whole blood assay uses fresh human blood (which contains a variety of Toll receptors) and measures the total release of TNF-a by ELISA [26]. Cells were stimulated with defined TLR agonists for TLR2; PolyIC for TLR3; E. coli Re595 LPS for TLR4; R848 for TLR7; and CpG Oligo (2 lM) for TLR9), and the production of TNF-a was quantified (Fig. 7). Even when
Scheme 1. The structures of oligosaccharides 1 and 2. Oligosaccharide 1, R ¼ a-Fuc3N (P); oligosaccharide 2, R ¼ a-Glc (C).
(cid:1) FEBS 2004
Analysis of Spirochaeta aurantia glycolipid LGLB (Eur. J. Biochem. 271) 4691
Table 2. NMR data for compounds LGLB, 1 and 2. Data refer to both compounds, except where indicated. N-Acetyl at I2: H-2/C-2 2.12/23.8, C-1176.2 p.p.m.
4 5 (5eq) 6 (5ax) Unit, compound 1 2 3 6b
3.90 A, a-Man
B, a-Fuc3N
P, a-Fuc3N (1)
C, a-Glc (2) 3.86 3.90 61.5 1.27 16.6 1.25 16.6 3.86 62.4 D, a-GalA 175.6 E, a-GalA6Asp 5.71 99.8 5.58 98.6 5.58 98.6 5.55 99.6 5.32 96.5 5.20 100.8 170.6 F, a-GalA 176.0 G, a-GlcA (1) 5.18 99.3 5.19 101.6 177.2 G, a-GlcA (2) 5.19 101.6 177.2 I, a-GalNAcA 176.0
1H 13C 1H 13C 1H 13C 1H 13C 1H 13C 1H 13C 1H 13C 1H 13C 1H 13C 1H 13C 1H 104.8 1H 104.4 1H 13C 1H 13C 1H 13C 1H 13C 1H
K, b-GlcA 13C L, b-GlcA (1) 13C L, b-GlcA (2) 5.11 99.3 4.80 74.7 4.81 74.9 4.81 104.4 M, b-Xyl 4.62 176.4 3.98 106.5 N, b-Gal 4.55 3.88 104.1 O, b-Xyl 4.47 3.88 62.3 3.98 106.6 3.92 67.7 4.02 69.4 4.01 69.4 3.47 70.5 4.69 79.4 4.70 79.4 4.50 80.3 3.83 77.3 3.82 77.8 4.69 77.6 3.91 78.0 3.83 78.1 3.83 77.4 3.72 70.5 4.29 66.4 3.69 70.5 3.72 74.1 4.18 67.7 4.16 67.7 3.78 73.0 4.65 72.4 5.05 72.2 4.79 72.8 4.30 73.7 4.30 73.7 4.76 72.9 3.90 176.3 3.77 176.4 3.74 78.1 3.33 66.4 3.80 76.2 3.33 66.4 Asp 4.12 81.2 4.01 66.3 3.99 66.3 3.59 73.0 4.22 68.7 4.12 68.8 3.89 68.9 3.69 73.1 3.69 73.1 4.46 49.8 3.50 77.4 3.34 77.5 3.34 74.9 3.39 74.4 3.72 70.4 3.28 73.9 4.42
13C 1H
13C 1H
178.8 178.8 Q, Gro, 1 and 2 52.3 4.03
13C
71.7 5.32 Q, Gro, LGLB
high concentrations of LGLB were added, no production of TNF-a was detected, showing that this large glycolipid cannot stimulate TLR2, -3, -4, -7 or -9.
3.85 4.00 72.1 3.84 3.84 69.8 72.0 3.99 71.5 3.68 53.6 3.65 53.6 3.76 74.0 4.23 79.3 4.20 79.4 4.21 69.6 4.06 75.0 4.06 75.0 4.10 77.3 3.86 77.4 3.75 77.4 3.75 77.5 3.46 77.3 3.91 78.6 3.42 77.1 2.93 2.93 39.1 3.68 3.76 63.7 4.15 4.50 64.8
Discussion
molecule were detected in patients with Lyme disease. A diacyl glycerol anchor has also been purposed for the glycolipids of T. denticola, T. maltophilum, and T. brenn- aborense [6,12]. A glycolipid identified in T. pectinovorum contained glycerol, and the majority of fatty acids were branched, although on the basis of detection of Kdo in this material, the authors designated it LPS. A diacyl glycerol anchor may substitute for lipid A, an observa- tion supported by the absence of any homologs to genes involved in lipid A biosynthesis in the completed ge- nomes of B. burgdorferi, T. pallidum or T. denticola [9,10,33].
All of the treponemal glycolipids identified have either fully saturated or branched fatty acids, in contrast to the
Although some structural information has been obtained from other spirochetes, the complete elucidation of the LGLB from S. aurantia represents the first complete structure of a large glycolipid from these bacteria. The dodecasaccharide LGLB is anchored by a diacyl glycerol. A glycolipid containing a single sugar, BbGL-II, and also anchored on a glycerol, has been identified in B. burg- dorferi [13]. It is surface localized, and antibodies to this
4692 E. Vinogradov et al. (Eur. J. Biochem. 271)
(cid:1) FEBS 2004
1180.25
1
1114.29
1000
1 100
1300
1400
1200
1197.25
2
1189.28
1180.30
1116.27
1131.28
1000
1100
1300
1400
1200
m/z
613.16
B
468.09
556.15
K
F
672.67
146.08
732.18
503.63
I
D
N
Q
L
O
M
C -G - A, NH3
648.20
218.06
789.21
714.18
292.06
824.24
1006.27
321.08
1199.29
339.09
663.67
807.22
1023.26
1182.24
376.09
1344.37
1436.42
1744.53
1876.56
1612.48
200
400
600
800
1000
1200
1400
1600
1800
2000
m/z
Fig. 5. CE-MS spectra of oligosaccharides 1 and 2.
Fig. 6. MS/MS spectrum obtained from a doubly charged ion at m/z 1197.25 of oligosaccharide 2.
(cid:1) FEBS 2004
Analysis of Spirochaeta aurantia glycolipid LGLB (Eur. J. Biochem. 271) 4693
40
35
30
25
20
15
) L m / g n ( α - F N T
10
5
0
No
Zymosan HKSA
PolyIC Re LPS R848
CpG
LGLB
size as
(HKSA)
Although there is no similarity at a structural level to LPS, studies investigating treponemal glycolipids have shown that most are able to gel LAL and stimulate Toll-like receptors [12,20], suggesting that at a functional level they possess some similarity. LGLB was able to gel LAL, but did not stimulate any TLR examined: this is an unusual situation, paralleled in the spirochete litera- ture only by the inability of the Borrelia glycolipids to activate TLR2 or -4 [13]. TNF-a release was measured following the exposure of human mononuclear cells to two different GSLs from S. paucimobilis: the mono- glycosylated GSL-1, and the tetraglycosylated GSL-4A. GSL-1 was unable to activate the release of monokines, in contrast to the larger GSL-4A, although induction was still 10 000-fold below that of the LPS standard [44]. While this appears to be similar to the situation with the monoglycosylated BbGL-II, the inability of LGLB to stimulate TNF-a release precludes the only explanation for the difference in biological activity observed with GSLs.
et al. unsaturated acyl group of BbGL-II. Schultz indicated that the presence of fatty acid branching in T. denticola is analogous to adaptations in Gram-positive bacteria to alter membrane fluidity [12]. Gram-negative bacteria are known to modify the degree of saturation in their fatty acids to modulate membrane fluidity [34,35]. LGLB contained both unsaturated and branched fatty acids (i.e. C14:0, the only spirochete iC15:0, C16:1), glycolipid identified, to date, with both of these modi- fications, suggesting LGLB may form highly fluid mem- branes.
S. aurantia LGLB comprises 15% lipid by mass, corres- ponding well with the proportion of fatty acids in the glycolipid OML521 (10.7%) from T. denticola, a glycolipid that is also estimated to be similar in size to Ra LPS [12]. Ra LPS is the minimum LPS unit required for efficient and proper folding, and functioning, of porin [36]. T. denticola and S. aurantia possess two of the largest porins yet discovered in Gram-negative bacteria: given the absence of LPS in these bacteria, OML251 and LGLB may function in place of Ra LPS, and contribute to the folding or stabilization of porin [37,38].
While S. aurantia stains Gram-negative and possesses an outer membrane containing porin, phylogenetically it is not closely related to the bacterial phylum (Proteobacte- ria) that contains the typical Gram-negative cells, such as E. coli. Other nonproteobacterial organisms in which glycolipids replace LPS include Chloroflexus aurantiacus and Fibrobacter succinogenes. The former bacterium is thought to contain outer membrane galactolipids [39], while the latter contains a low molecular mass glycolipid with glycerol anchor and many charged groups in the oligosaccharide part, which makes it, in overall design, similar to S. aurantia LGLB. Interestingly F. succinogenes also has a capsular polysaccharide with a lipid anchor [40].
Even within the Proteobacteria, one finds examples where LPS has been replaced by glycolipids. Sphingomonas pau- cimobilis and Novosphingobium capsulatum contain glyco- sphinogolipids (GSLs) [41–43] in lieu of LPS.
Another oral spirochete implicated in periodontal dis- ease, T. medium, contains the glycolipid, Tm-Gp, which abrogates TLR activation through interactions with LPS-binding protein (LBP) and CD14, two important components of TLR-mediated innate immunity [45]. The blocking by Tm-Gp was dependent on the lipid portion of the molecule, but whether S. aurantia LGLB would block a TLR response is unknown. Structural studies of Tm-Gp have focused on a tetrasaccharide repeating unit, likened by Asai and colleagues to the repeating unit of the LPS O-antigen [11]. The characteristic laddering pattern on SDS/ PAGE suggests that Tm-Gp is different from LGLB, although they both contain an aspartic acid residue. The structures of the bioactive portion of Tm-Gp, and of the other treponemal glycolipid TLR agonists, need to be elucidated to begin to identify possible motifs involved in modulating TLR activity. This is especially interesting when one realizes that the existing literature does not contain any direct demonstration of a ligand-type interaction between a TLR and any glycoconjugate, LPS or otherwise. LPS has been shown, however, to bind LBP [46]. Interestingly, a decrease in the fluidity of Re LPS, instigated by a Zn2+- induced increase in acyl chain order, elevated the production of TNF-a from human monocytes. The increase in acyl chain order increased the bond strength between Re LPS and LBP, and was thought to increase the transport of the LPS to the target membrane. LBP is an important precursor in the TLR-dependent release of TNF-a and has been shown to interact with both the T. maltophilum and T. brennaborense glycolipids to enhance their ability to stimulate TLRs [6]. It is tempting to speculate that the highly disordered acyl chains of LGLB could abrogate the interaction with LBP and prevent any release of TNF-a in the whole blood assay for TLR activation. Specific struc- tural entities of LPS, producing certain biological effects, have been extensively studied given the central role of this molecule in pathogenesis and vaccine development. Char- acterization of any biological activity of spirochete glyco- lipids is important for similar reasons, especially in the case of B. burgdorferi BbGL-II, given the difficulties in develop- ing an effective proteinaceous vaccine targeting this organ- ism [13,47].
Fig. 7. Tumour necrosis factor-a (TNF-a) production through activation of Toll-like receptors (TLR) in the presence of different agonists. Con- trols for different TLR were as follows: zymosan and heat-killed Staphylococcus aureus for TLR2; PolyIC for TLR3; Escherichia coli Re595 lipopolysaccharide (LPS) for TLR4; R848 for TLR7; and CpG Oligo for TLR9. Error bars represent the standard deviation of cellular activation experiments performed in triplicate.
4694 E. Vinogradov et al. (Eur. J. Biochem. 271)
(cid:1) FEBS 2004
Acknowledgements
This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to A.M.K. 14. Ben-Menachem, G., Kubler-Kielb, J., Coxon, B., Yergey, A. & Schneerson, R. (2003) A newly discovered cholesteryl galactoside from Borrelia burgdorferi. Proc. Natl Acad. Sci. USA 100, 7913– 7918. C.J.P. was the recipient of an NSERC studentship.
15. Triantafilou, M. & Triantafilou, K. (2002) Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends Immunol. 23, 301–304.
References
1. Paster, B.J., Dewhirst, F.E., Weisburg, W.G., Tordoff, L.A., Fraser, G.J., Hespell, R.B., Stanton, T.B., Zablen, L., Mandelco, L. & Woese, C.R. (1991) Phylogenetic analysis of the spirochetes. J. Bacteriol. 173, 6101–6109. 16. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C.X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B. & Beutler, B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in TLR 4 gene. Science 282, 2085–2088.
2. Breznak, J.A. & Canale-Parola, E. (1969) Spirochaeta aurantia, a pigmented, facultatively anaerobic spirochete. J. Bacteriol. 97, 386–395. 17. Hirschfeld, M., Ma, Y., Weis, J.H., Vogel, S.N. & Weis, J.J. (2000) Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2. J. Immunol. 165, 618–622.
3. Breznak, J.A. & Canale-Parola, E. (1975) Morphology and phy- siology of Spirochaeta aurantia strains isolated from aquatic habitats. Arch. Microbiol. 105, 1–12.
4. Lilburn, T.G., Kim, K.S., Ostrom, N.E., Byzek, K.R., Leadbetter, J.R. & Breznak, J.A. (2001) Nitrogen fixation by symbiotic and free-living spirochetes. Science 292, 2495–2498. 18. Hirschfeld, M., Weis, J.J., Toshchakov, V., Salkowski, C.A., Cody, M.J., Ward, D.C., Qureshi, N., Michalek, S.M. & Vogel, S.N. (2001) Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69, 1477–1482.
5. Asai, Y., Jinno, T. & Ogawa, T. (2003) Oral treponemes and their outer membrane extracts activate human gingival epithelial cells through toll-like receptor 2. Infect. Immun. 71, 717–725. 19. Girard, R., Pedron, T., Uematsu, S., Balloy, V., Chignard, M., Akira, S. & Chaby, R. (2003) Lipopolysaccharides from Legio- nella and Rhizobium stimulate mouse bone marrow granulocytes via Toll-like receptor 2. J. Cell Sci. 116, 293–302.
6. Schro¨ der, N.W., Opitz, B., Lamping, N., Michelsen, K.S., Za¨ hringer, U., Go¨ bel, U.B. & Schumann, R.R. (2000) Involve- ment of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Trepo- nema glycolipids. J. Immunol. 165, 2683–2693. 7. Sela, M.N. (2001) Role of Treponema denticola in periodontal 20. Opitz, B., Schro¨ der, N.W., Spreitzer, I., Michelsen, K.S., Kirsch- ning, C.J., Hallatschek, W., Za¨ hringer, U., Hartung, T., Go¨ bel, U.B. & Schumann, R.R. (2001) Toll-like receptor-2 mediates Treponema glycolipid and lipoteichoic acid-induced NF-jB translocation. J. Biol. Chem. 276, 22041–22047. diseases. Crit Rev. Oral Biol. Med. 12, 399–413.
21. Erridge, C., Pridmore, A., Eley, A., Stewart, J. & Poxton, I.R. (2004) Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachomatis and Pseudomonas aeruginosa signal via Toll-like receptor 2. J. Med. Microbiol. 53, 735–740.
8. Werts, C., Tapping, R.I., Mathison, J.C., Chuang, T.H., Krav- chenko, V., Saint, G.I., Haake, D.A., Godowski, P.J., Hayashi, F., Ozinsky, A., Underhill, D.M., Kirschning, C.J., Wagner, H., Aderem, A., Tobias, P.S. & Ulevitch, R.J. (2001) Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat. Immunol. 2, 346–352. 22. Darveau, R.P. & Hancock, R.E. (1983) Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseu- domonas aeruginosa and Salmonella typhimurium strains. J. Bac- teriol. 155, 831–838.
23. Tsai, C.M. & Frasch, C.E. (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Bio- chem. 119, 115–119.
9. Fraser, C.M., Norris, S.J., Weinstock, G.M., White, O., Sutton, G.G., Dodson, R., Gwinn, M., Hickey, E.K., Clayton, R., Ket- chum, K.A., Sodergren, E., Hardham, J.M., McLeod, M.P., Salzberg, S., Peterson, J., Khalak, H., Richardson, D., Howell, J.K., Chidambaram, M., Utterback, T., McDonald, L., Artiach, P., Bowman, C., Cotton, M.D. & Venter, J.C. (1998) Complete genome sequence of Treponema pallidum, the syphilis spirochete. Science 281, 375–388. 24. Lesse, A.J., Campagnari, A.A., Bittner, W.E. & Apicella, M.A. (1990) Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate- polyacrylamide gel electrophoresis. J. Immunol. Methods 126, 109– 117.
25. Gu, X.X., Chen, J., Barenkamp, S.J., Robbins, J.B., Tsai, C.M., Lim, D.J. & Battey, J. (1998) Synthesis and characterization of lipooligosaccharide-based conjugates as vaccine candidates for. Moraxella (Branhamella) catarrhalis. Infect. Immun. 66, 1891– 1897.
10. Fraser, C.M., Casjens, S., Huang, W.M., Sutton, G.G., Clayton, R., Lathigra, R., White, O., Ketchum, K.A., Dodson, R., Hickey, E.K., Gwinn, M., Dougherty, B., Tomb, J.F., Fleischmann, R.D., Richardson, D., Peterson, J., Kerlavage, A.R., Quackenbush, J., Salzberg, S., Hanson, M., van Vugt, R., Palmer, N., Adams, M.D., Gocayne, J. & Venter, J.C. (1997) Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390, 580– 586.
26. Tapping, R.I., Akashi, S., Miyake, K., Godowski, P.J. & Tobias, P.S. (2000) Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor for Escherichia and Salmonella lipopoly- saccharides. J. Immunol. 165, 5780–5787.
11. Hashimoto, M., Asai, Y., Jinno, T., Adachi, S., Kusumoto, S. & Ogawa, T. (2003) Structural elucidation of polysaccharide part of glycoconjugate from Treponema medium ATCC 700293. Eur. J. Biochem. 270, 2671–2679. 27. Kropinski, A.M., Ghiorse, W.C. & Greenberg, E.P. (1988) The intracellular polyglucose storage granules of Spirochaeta aurantia. Arch. Microbiol. 150, 289–295.
12. Schultz, C.P., Wolf, V., Lange, R., Mertens, E., Wecke, J., Nau- mann, D. & Zahringer, U. (1998) Evidence for a new type of outer membrane lipid in oral spirochete Treponema denticola. Func- tioning permeation barrier without lipopolysaccharides. J. Biol. Chem. 273, 15661–15666. 28. Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J. & Carlo, D.J. (1978) A new and improved microassay to determine 2-keto-3- deoxyoctonate in lipopolysaccharide of Gram-negative bacteria. Anal. Biochem. 85, 595–601.
13. Schro¨ der, N.W., Schombel, U., Heine, H., Go¨ bel, U.B., Za¨ hrin- ger, U. & Schumann, R.R. (2003) Acylated cholesteryl galactoside as a novel immunogenic motif in Borrelia burgdorferi sensu stricto. J. Biol. Chem. 278, 33645–33653. 29. Kropinski, A.M., Lewis, V. & Berry, D. (1987) Effect of growth temperature on the lipids, outer membrane proteins, and lipo- polysaccharides of Pseudomonas aeruginosa PAO. J. Bacteriol. 169, 1960–1966.
(cid:1) FEBS 2004
Analysis of Spirochaeta aurantia glycolipid LGLB (Eur. J. Biochem. 271) 4695
30. Vinogradov, E.V., Kaca, W., Rozalski, A., Shashkov, A.S., Cedzynski, M., Knirel, Y.A. & Kochetkov, N.K. (1991) Structural and immunochemical studies of O-specific polysaccharide of Proteus vulgaris 5/43 belonging to OX19 group (O-variants). Eur. J. Biochem. 200, 195–201.
for the identification of 38. Kropinski, A.M., Parr, T.R. Jr , Angus, B.L., Hancock, R.E., Ghiorse, W.C. & Greenberg, E.P. (1987) Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia. J. Bacteriol. 169, 172–179. 39. Meissner, J., Krauss, J.H., Ju¨ rgens, U.J. & Weckesser, J. (1988) Absence of a characteristic cell wall lipopolysaccharide in the phototrophic bacterium Chloroflexus aurantiacus. J. Bacteriol. 170, 3213–3216.
40. Vinogradov, E., Egbosimba, E.E., Perry, M.B., Lam, J.S. & Forsberg, C.W. (2001) Structural analysis of the carbohydrate components of the outer membrane of the lipopolysaccharide- lacking cellulolytic ruminal bacterium Fibrobacter succinogenes S85. Eur. J. Biochem. 268, 3566–3576.
31. Thibault, P., Li, J., Martin, A., Richards, J.C., Hood, D.W. & Moxon, E.R. (1997) Electrophoretic and mass spectrometric strategies lipopolysaccharides and immunodeterminants in pathogenic strains of Haemophilis influ- enzae; application to clinical isolates. In Mass Spectrometry in Biology and Medicine (Burlingame, A.L., Baldwin, M.A. & Carr, S.A., eds), pp. 439–462. Humana Press, Totowa, NJ, USA. 32. Hansen, L.A., Poulsen, O.M. & Wurtz, H. (1999) Endotoxin potency in the A549 lung epithelial cell bioassay and the limulus amebocyte lysate assay. J. Immunol. Methods 226, 49–58.
41. Kawasaki, S., Moriguchi, R., Sekiya, K., Nakai, T., Ono, E., Kume, K. & Kawahara, K. (1994) The cell envelope structure of the lipopolysaccharide-lacking gram-negative bacterium Sphingo- monas paucimobilis. J. Bacteriol. 176, 284–290.
42. Kawahara, K., Seydel, U., Matsuura, M., Danbara, H., Rietschel, E.T. & Zahringer, U. (1991) Chemical structure of glyco- sphingolipids isolated from Sphingomonas paucimobilis. FEBS Lett. 292, 107–110.
43. Kawahara, K., Moll, H., Knirel, Y.A., Seydel, U. & Za¨ hringer, U. (2000) Structural analysis of two glycosphingolipids from the lipopolysaccharide-lacking bacterium Sphingomonas capsulata. Eur. J. Biochem. 267, 1837–1846. 33. Seshadri, R., Myers, G.S., Tettelin, H., Eisen, J.A., Heidelberg, J.F., Dodson, R.J., Davidsen, T.M., DeBoy, R.T., Fouts, D.E., Haft, D.H., Selengut, J., Ren, Q., Brinkac, L.M., Madupu, R., Kolonay, J., Durkin, S.A., Daugherty, S.C., Shetty, J., Shvarts- beyn, A., Gebregeorgis, E., Geer, K., Tsegaye, G., Malek, J., Ayodeji, B., Shatsman, S., McLeod, M.P., Smajs, D., Howell, J.K., Pal, S., Amin, A., Vashisth, P., McNeill, T.Z., Xiang, Q., Sodergren, E., Baca, E., Weinstock, G.M., Norris, S.J., Fraser, C.M. & Paulsen, I.T. (2004) Comparison of the genome of the oral pathogen Treponema denticola with other spirochete genomes. Proc. Natl Acad. Sci. USA 101, 5646–5651.
from Sphingomonas paucimobilis
[Review] 44. Krziwon, C., Za¨ hringer, U., Kawahara, K., Weidemann, B., Kusumoto, S., Rietschel, E.T., Flad, H.D. & Ulmer, A.J. (1995) Glycosphingolipids induce monokine production in human mononuclear cells. Infect. Immun. 63, 2899–2905. 34. Heipieper, H.J., Meinhardt, F. & Segura, A. (2003) The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a [48 refs]. FEMS unique stress adaptive mechanism. Microbiol. Lett. 229, 1–7.
45. Asai, Y., Hashimoto, M. & Ogawa, T. (2003) Treponemal gly- coconjugate inhibits Toll-like receptor ligand-induced cell activa- tion by blocking LPS-binding protein and CD14 functions. Eur. J. Immunol. 33, 3196–3204. 35. Sakamoto, T. & Murata, N. (2002) Regulation of the desaturation of fatty acids and its role in tolerance to cold and salt stress. Curr. Opin. Microbiol. 5, 208–210.
46. Tobias, P.S., Soldau, K., Gegner, J.A., Mintz, D. & Ulevitch, R.J. (1995) Lipopolysaccharide binding protein-mediated complexa- tion of lipopolysaccharide with soluble CD14. J. Biol. Chem. 270, 10482–10488. 36. Hagge, S.O., de Cock, H., Gutsmann, T., Beckers, F., Seydel, U. & Wiese, A. (2002) Pore formation and function of phosphoporin PhoE of Escherichia coli are determined by the core sugar moiety of lipopolysaccharide. J. Biol. Chem. 277, 34247–34253.
47. Hanson, M.S. & Edelman, R. (2003) Progress and controversy surrounding vaccines against Lyme disease. Expert Rev. Vaccines 2, 683–703. 37. Egli, C., Leung, W.K., Muller, K.H., Hancock, R.E. & McBride, B.C. (1993) Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola. Infect. Immun. 61, 1694–1699.
