Structural analysis of Francisella tularensis lipopolysaccharide Evgeny Vinogradov, Malcolm B. Perry and J. Wayne Conlan Institute for Biological Sciences, National Research Council, Ottawa, Canada The structure of the lipid A and core region of the lipo- polysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a b-GlcN-(1–6)- GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O-1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O-3 and O-4 of b-GlcN, and no substituent at O-4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH 4 reduced LPS had the fol- lowing structure: where D-GalNA-(1–3)-b-QuiNAc represents a modified fragment of the O-chain repeating unit. Two shorter oligo- saccharides lacking the O-chain fragment were also identified. A minor amount of the disaccharide b-GlcN-(1–6)-a-GlcN- 1-P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end.The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2-N-(3-O- acyl 4 -acyl 2 )-b-GlcN-(1–6)-2-N-acyl 1 )3-O-acyl 3 -GlcN where acyl 1 ,acyl 2 and acyl 3 are 3-hydroxyhexadecanoic or 3-hy- droxyoctadecanoic acids, acyl 4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH-1 of the reducing end glucosamine, and OH-3 and OH-4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure. Keywords: Francisella tularensis; lipopolysaccharide; core; lipid A. Francisella tularensis is a Gram-negative bacterium which causes tularemia, a severe and often fatal disease of humans and other mammals [1]. The bacterium is an intracellular pathogen and therefore cell-mediated, rather than humoral, immunity is thought to be required to combat it [1–3]. However, it has also been shown that antibodies directed against the LPS of F. tularensis can ameliorate the course of tularemia [4,5]. Additionally, F. tularensis LPS possesses unusual biological properties that also presumably influence the disease process. For instance, F. tularensis LPS lacks endotoxicity and is a poor inducer of proinflammatory cytokines [6]. On the other hand, it has been shown recently that subimmunogenic doses of LPS derived from F. tula- rensis live vaccine strain (LVS) can elicit an unusual, and apparently specific, anti-Francisella resistance that relies on the actions of interferon-gamma and B-cells, but not antibodies, for its expression [7]. Knowledge of the fine structure of F. tularensis LPS will be needed to explain these biological activities. In previous studies [8] we have described the structure of the O-antigen produced by F. tularensis ATCC 29684, which proved to be identical to the structure of strain 15 [9]. The present study focuses on the structure of the lipid A and core region of the F. tularensis LPS ATCC 29684. EXPERIMENTAL PROCEDURES Lipopolysaccharide isolation F. tularensis LVS (ATCC 29684) was grown to D 600  1.1 in a 40-L batch in Trypticase soy broth containing 0.1% (w/v) cysteine/HCl and 0.025% (w/v) ferric pyrophosphate. Cells were killed by the addition of phenol (final concentra- tion 2%, v/v), and harvested by continuous centrifugation at 62 000 g (yield  1gwetwt.ÆL )1 ). The saline-washed cells (250 g wet wt.) were extracted by stirring with 400 mL 50% (v/v) aqueous phenol at 65 °C for 15 min [10]. The cooled extract was diluted with water (2 vol.) and the cleared extract was dialyzed against tap water until free from phenol. The dialyzed retentate was dissolved in 90 mL NaOAc (0.02 M , pH 7.0) and was treated sequentially with RNase, DNase and proteinase K (37 °C, 2 h each). The ensuing mixture was Correspondence to Evgeny Vinogradov, Institute for Biological Sciences, National Research Council, 100 Sussex Dr, K1A 0R6 Ottawa ON, Canada. Fax: + 1 613 952 90 92, Tel.: + 1 613 990 0832, E-mail: evguenii.vinogradov@nrc.ca Abbreviations: GalA, galacturonic acid; GalNA, galactosaminuronic acid; D-GalNA, 2,4-dideoxy-2-amino-b- L -threo-hex-4-eno-pyranosyl; HPAEC, high-performance anion-exchange chromatography; LPS, lipopolysaccharide; Kdo, 3-deoxy- D -manno-octulosonic acid; QuiN, 2-amino-2,6-dideoxyglucose. (Received 3 July 2002, revised 2 October 2002, accepted 22 October 2002) Eur. J. Biochem. 269, 6112–6118 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03321.x cleared by low speed centrifugation (3000 g), and subjected to sequential ultracentrifugation at 27 000 g (precipitate designated K27), and 60 000 g (precipitate K60), both for 10 h at 4 °C. The precipitated gels were dissolved in distilled water and lyophilized. The K27 fraction (1.72 g) contained LPS contaminated with  40% (w/w) of an amylopectin- like D -glucan. The K60 fraction (840 mg) was essentially pure S-type LPS and was used in subsequent studies. NMR spectroscopy and general methods 1 H- and 13 C-NMR spectra of lipid A were recorded using a Varian Inova 500 spectrometer in CDCl 3 –CD 3 OD (3 : 1, v/v) or in CDCl 3 -CD 3 OH (3 : 1, v/v). Solutions were at 25 °C and referenced to the residual chloroform signal ( 1 H 7.26 p.p.m.) and MeOH ( 13 C 49.15 p.p.m.); spectra of all other compounds were recorded at 25 °CinD 2 Oand referenced to acetone (d H 2.225 p.p.m., d C 31.45 p.p.m.). Varian standard pulse sequences COSY, TOCSY (mixing time 100 ms), ROESY (mixing time 200 ms), HSQC and gHMBC (optimized for 5 Hz coupling constant) were used. Electrospray mass spectra were obtained using a Micromass Quattro spectrometer in 50% (v/v) MeCN with 0.2% (v/v) HCOOH at a flow rate of 15 lLÆmin )1 with direct injection in negative mode. MALDI-TOF mass spectra were recor- ded in positive mode with a Bruker-Reflex III spectrometer (Bruker-Franzen Analytik, Bremen, Germany) in both linear and reflection TOF configurations at an acceleration voltage of 20 kV and delayed ion extraction. The samples were dissolved in aqueous triethylamine (0.07 M )ata concentration of 2 lgÆlL )1 .OnelL of the sample was then mixedwith1lLof0.5 M matrix solution of recrystal- lized 2,5-dihydroxybenzoic acid (Aldrich, Deisenhofen, Germany) in methanol containing 0.1% (v/v) trifluoroacetic acid. Aliquots of 0.5 lL were deposited on a metallic sample holder and analyzed immediately after drying in a stream of air. The instrument was calibrated externally with similar compounds of known structure. The mass spectra shown are the sum of at least 50 laser shots. GC was performed on an HP1 column (30 m · 0.25 mm) using an Agilent 6850 chromatograph fitted with a flame ionization detector, or on a Varian Saturn 2000 ion-trap GC/MS instrument. Lipid A isolation LPS (200 mg) was hydrolysed with 5% (v/v) AcOH (100 °C, 4 h) and the precipitated product was collected by centri- fugation at 3000 g and suspended in 2% (v/v) MeOH in CHCl 3 . It was applied to a silica gel column (2 · 8cm),then washed sequentially with 2%, 5%, 10% and 20% (v/v) MeOH in CHCl 3 . Clean lipid A (20 mg) was recovered from the 10% (v/v) MeOH eluate; 5% and 20% (v/v) MeOH contained minor amounts of lipid-like components. Fatty acid analysis Lipid (2 mg) was dissolved in CHCl 3 –MeOH (3 : 1, v/v, 1 mL total volume) and 1 M MeONa in MeOH (0.2 mL) was added. The mixture was kept for 24 h at 25 °C, acidified with trifluoroacetic acid, evaporated and extracted with hexane. Samples of lipid containing hexane extract and hexane-insoluble material were treated with 1 M HCl in MeOH (1 mL, 100 °C, 4 h), dried, and analysed by GC and GC/MS. Hydrofluoric acid cleavage Lipid A sample (10 mg) was stirred with 48% (v/v) hydrofluoric acid in a volume of 1.0 mL in a poly(ethylene) vial at 20 °C for 24 h, then dried in vacuum dessicator over NaOH. The product was analysed by NMR and MS without purification. LPS reduction LPS (150 mg) was dissolved in water (100 mL), then 300 mg NaBH 4 and a drop of octanol to prevent foaming were added. The mixture was kept at room temperature for 24 h, then dialysed against water. After lyophylization 120 mg of reduced LPS was recovered. Alkaline deacylation Two LPS samples (80 mg each) and NaBH 4 -reduced LPS (80 mg) were dissolved in 4 M KOH (4 mL each). To one of the LPS samples 100 mg NaBH 4 were added, kept overnight at 100 °C and neutralized with 2 M HCl. Precipitated mater- ial was removed by centrifugation at 3000 g and the solutions were passed through SepPak C18 cartridges (washed with MeOH and water before use) and applied to a Sephadex G50 column. The fractions were analysed by NMR spectroscopy and ESI/MS, and those containing core oligosaccharides were separated by HPAEC in a gradient of 0.1 M NaOH (A) to 1 M AcONa in 0.1 M NaOH (B), using 5–50% of B. Products were desalted on a Sephadex G15 column. Hydrazine treatment LPS (100 mg) or lipid precipitate from AcOH hydrolysis (30 mg) were dissolved in anhydrous hydrazine (3 mL) and kept at 40 °C for 1 h. Samples were transferred to plastic Petri dishes to provide a large surface for evaporation and hydrazine was removed in a vacuum dessicator over sulfuric acid. Products were dissolved in water, precipitates were removed by centrifugation at 5000 g, and the solutions were dried and analysed by NMR spectroscopy. Sample obtained from lipid precipitates contained mostly a b-glucan 5,which was purified by ion exchange chromatography on a HiTrap Q column (Pharmacia) in water (A) to 1 M NaCl (B), with a gradient from 0–100% NaCl. Sample prepared from LPS was fractionated on Sephadex G50 column to give a-(1–6)- glucan, amylopectin, b-glucan 5, and fragments of the O-chain. RESULTS Lipid A was liberated from F. tularensis LVS (ATCC 29684) LPS by acetic acid hydrolysis. This LPS was more stable to acetic acid hydrolysis than LPSs form most other bacteria, and the lipid A moiety could only be cleaved from the rest of the molecule using hot 5% (v/v) acetic acid rather than the usual 1–2% concentration. Lipid A was then purified by conventional silica gel chromatography. Com- parison of the 1 H-NMR spectra of the unfractionated lipid A and chromatographically fractionated samples indicated that the fraction eluted with 10% (v/v) MeOH in CHCl 3 contained the major component. It was used in further studies as Ôlipid AÕ. Ó FEBS 2002 Francisella tularensis lipopolysaccharide (Eur. J. Biochem. 269) 6113 Fatty acid analysis of the purified lipid A showed the presence of C14:0, C16:0, C16:0(3-OH), and C18:0(3-OH) straight chain acids in the ratio of 1 : 0.2 : 1.6 : 4. In order to distinguish between ester- and amide-linked acids, the lipid A was treated with MeONa for O-deacylation, released acids were extracted into hexane, and both the hexane extract and the residual material were analyzed by GC after methanolysis. C14:0 and C16:0 acids were found to be completely released following O-deacylation, whereas C16:0(3-OH) and C18:0(3-OH) were distributed in both fractions and were thus present in both ester- and amide- linked forms. NMR analysis of the lipid using 2D techniques (Table 1, Fig. 1) led to the identification of b-GlcN-(1–6)-GlcN backbone disaccharide, carrying four acyl residues. GlcN residue A had unsubstituted hydroxyl group at C-1, and was mostly present in an a-pyranose form. Acyl 1 ,acyl 2 and acyl 3 residues had hydroxy or acyloxy groups at C-3 ( 13 C signals of C-3 at 69.0–72.4 p.p.m.), while acyl 4 had no substituents. The signals of acyl chains could only be identified up to C-4, H-4, because of the overlap of the remaining signals. Distribution of the acyl residues was deduced from NOE correlations between amide protons and the H-2 of acyl residues, and from HMBC correlations between C-1 of acyl groups and protons at the acylation site (Fig. 1). NOE between protons A2 and acyl 1 -2, and between B2and acyl 2 -2 indicated that GlcN A is N-acylated with acyl 1 ,and GlcN B is N-acylated with acyl 2 . All acyl C-1 signals were identified from H-2:C-1 HMBC correlations. C-1 of acyl 2 gave HMBC correlation to H-2 of GlcN B; C-1 of acyl 4 showed correlation to H-3 of acyl 2 . C-1 Signals of acyl 1 and acyl 3 overlap, but because acyl 1 can be identified as the acylating amino group of GlcN A on the basis of NOE correlation, HMBC correlation at 5.00 (H) to 173.9 (C) p.p.m. can only be explained as resulting from the acylation of A3withacyl 3 . O-Acylation of GlcN A at O-3 and of acyl 2 at O-3 agreed with the low-field position of the correspond- ing proton signals (Table 1). These results identify the disaccharide backbone structure and acylation pattern except for the length of the carbon chain of the acyl residues. Further information on the lipid A structure was obtained from MALDI mass spectra. The mass spectrum Table 1. NMR data for lipid A. Unit Nucleus 1 2(/2b or /NH) 3 4 5 6a 6b HMBC from C-1 A 1 H 4.92 3.94/7.09 5.00 3.34 3.88 3.56 3.89 13 C 92.0 52.8 75.0 69.6 71.2 69.7 B 1 H 4.32 3.41/7.62 3.34 3.20 3.13 3.56 3.69 13 C 102.5 56.9 75.6 71.5 76.7 62.4 A6 acyl 11 H 2.06/2.15 3.70 1.30 13 C 173.9 43.9 69.3 37.9 A2 acyl 21 H 2.28/2.33 5.01 1.46 1.14 13 C 172.9 42.2 72.4 34.9 26.0 B2 acyl 31 H 2.22/2.32 3.81 1.25 13 C 173.9 43.3 69.0 37.9 A3 acyl 41 H 2.14 1.43 1.14 13 C 175.4 35.2 25.7 30.1 acyl 2 H-3 Fig. 1. Fragments of HSQC and HMBC spectra of F. tularensis (ATCC 29684) lipid A. Signals of lipid with the a-anomeric form of GlcN A are labeled. 6114 E. Vinogradov et al.(Eur. J. Biochem. 269) Ó FEBS 2002 of the lipid A (Fig. 2) contained four major clusters of signals with the first peaks at m/z 1392.6, 1408.6, 1420.7 and 1436.7. These signals correspond to sodium and potassium adducts of two structural variants with masses of 1370.0 and 1398.1 Da. Since the nonhydroxylated acyl is known from GC analysis to be mostly C14:0, the remaining are two C18:0(3-OH) and one C16:0(3-OH) (1370.0 Da), or three C18:0(3-OH) residues (1398.1 Da), respectively. This was confirmed by the results of MALDI analysis of a lipid A sample treated with 48% (v/v) hydrofluoric acid. This treatment cleaved the glycosidic linkage between monosaccharide residues with- out affecting the acylation. The mass spectrum of the resulting mixture of units A and B contained peaks at m/z 694.82 (unit B with C14:0 and C18:0(3-OH) + Na + ), 738.9 Da (unit A with C16:0(3-OH) and C18:0 (3-OH) + Na + ), and 766.87 Da (unit A with two C18:0(3-OH) + Na + ). Minor peaks of unit A with two C16:0(3-OH) acyl residues (m/z 710.77), and of unit B with C16:0 and C18:0(3-OH) acyl residues (m/z 722.83 Da) were also observed. These results show that acyl 1 and acyl 3 can be C16:0(3-OH) or C18:0(3-OH), acyl 2 is mostly C18:0(3-OH); and acyl 4 is C14:0 with minor amount of C16:0. Combined NMR and MS evidence led to the proposed structure (Fig. 3), where acyl 1 ,acyl 2 and acyl 3 are 3-hydroxyhexadecanoic or 3-hydroxyoctadecanoic acids, acyl 4 is tetradecanoic or (minor) hexadecanoic acids. No core-related products were isolated from the AcOH hydrolysate of the LPS, probably because of a low content of rough variants of the LPS, and the presence of contaminants. Deacylation of the LPS with 4 M KOH led to four major products 1a, 2a, 3a and 4,isolatedby HPAEC. Compounds 1a, 2a and 3a contained an unidentified aglycon, derived from GlcN B by alkaline degradation. To avoid this degradation and confirm the absence of the substituent at O-1 of GlcN A,LPSwas reduced with NaBH 4 prior to alkaline treatment or treated with KOH in the presence of NaBH 4 . Both procedures led to the same products 1b, 2b and 3b,inwhichKdowas linked to )6)-b-GlcN-(1–6)-GlcN-ol. N-Acetyl group on the QuiN residue J in compounds 3a and 3b was exceptionally stable and was not removed under deacyla- tion conditions. NMR spectra of the products 1–4 were assigned (except for aglycon signals in 1a, 2a and 3a) using 2D techniques (Table 2, Fig. 4). Monosaccharides were identified on the basis of vicinal proton coupling constants and 13 CNMR chemical shifts. Anomeric configurations were deduced from the J 1,2 coupling constants and chemical shifts of H-1, C-1 and C-5 signals. The b-configuration of mannose residue F was confirmed by the observation of strong intraresidual NOE between H-1 and H-3, and between H-1 and H-5. Residue K is a product of an alkaline b-elimination of the O-4 substituent from a a-galactos- aminuronamide residue, present in the LPS O-chain [8,9]. Connections between monosaccharides were identified on the basis of NOE and HMBC correlations. The following NOEs were observed in the product 3b: B1A6, E1C5, E1C7, E1I1, F1E4, F1E6, I1E2, G1F2, G1F3, H1F2, H1G5, J1F4, J1F6andK1J3. Respective correlations, where applicable, were also observed in the smaller Fig. 2. MALDI mass spectrum of the purified lipid A. Fig. 3. Structure of the lipid A and its fragment, obtained after partial hydrolysis with 48% (v/v) HF. Acyl 1 and acyl 3 are C16:0(3-OH) or C18:0(3-OH), acyl 2 is mostly C18:0(3-OH), and acyl 4 is C14:0 with minor amount of C16:0. Ó FEBS 2002 Francisella tularensis lipopolysaccharide (Eur. J. Biochem. 269) 6115 oligosaccharides. Structures 1b, 2b and 3b were confirmed by ESI MS. In the structures 1a, 2a and 3a, aglycon R had mass of 101 Da, the same that was observed for the products of similar degradation of other LPSs [E. Vinogradov, M. B. Perry & J. W. Conlan, unpublished data]. The structure of this fragment was not understood. Together these data led to the structures presented in Fig. 5. Structures 1 and 2 represent LPS lacking O-chain, and structure 3 contains units K and J, corresponding to a part of the O-chain repeating unit. Thus compound 3 is derived from smooth LPS form. In an attempt to determine the base labile constituents of the LPS, anhydrous hydrazine treatment (40°, 1 h) of the LPS was performed. This experiment produced no explain- able results regarding LPS structure. O-Chain was depo- lymerized due to the presence of 4-substituted residues of galactosaminuronamide. Separation of the products on Fig. 4. 1 H NMR spectra of compounds 1a, 2a, 3a and 4. Spectrum of compound 1a was recorded at 30 °C to avoid overlap of water signal with F1 signal. Fig. 5. Structures of the oligosaccharides, obtained by KOH deacylation of F. tularensis LPS. Table 2. NMR data for the oligosaccharides 1-3. N-acetyl group signals in 3: H-2 1.89 ppm, C-2 23.2 ppm. Residue Nucleus 1 2 (3ax) 3 (3eq) 4 5 6 (6a) 7 (6b) 8a 8b C, 1,2,3 1 H 1.70 2.02 4.17 4.10 3.65 3.80 3.67 3.90 13 C 36.2 66.3 77.3 72.3 70.4 64.1 E, 1,2,3 1 H 5.13 4.26 4.01 3.93 4.00 3.74 3.74 13 C 100.3 78.0 69.5 76.5 72.5 60.5 F, 1 1 H 4.79 4.29 3.83 3.68 3.45 3.79 3.95 13 C 100.4 75.4 74.3 67.4 77.3 61.5 F, 2 1 H 4.86 4.40 4.01 3.93 3.48 3.80 3.95 13 C 100.1 73.1 79.1 67.7 76.9 61.0 F, 3 1 H 4.82 4.37 4.03 4.23 3.44 3.72 3.94 13 C 100.1 74.0 78.2 72.9 76.3 60.5 G, 2 1 H 5.36 3.62 3.59 3.37 3.80 3.71 3.91 13 C 100.1 71.8 73.8 70.3 73.5 61.5 G, 3 1 H 5.43 3.59 3.58 3.37 3.78 3.70 3.88 13 C 100.2 72.2 74.4 70.4 73.7 61.5 H, 1 1 H 5.57 3.53 4.15 3.99 4.30 3.66 3.66 13 C 96.7 51.9 67.2 68.7 71.6 61.2 H, 2 1 H 5.71 3.57 4.11 3.96 4.23 3.65 3.70 13 C 95.2 51.7 67.1 68.7 72.2 61.4 H, 3 1 H 5.65 3.56 4.13 3.97 4.24 3.64 3.68 13 C 95.5 51.8 67.1 68.7 72.4 61.5 I, 1,2,3 1 H 4.50 3.32 3.51 3.39 3.48 3.72 3.90 13 C 102.5 73.6 76.2 70.4 76.7 61.6 J 1 H 4.60 3.77 3.88 3.43 3.54 1.31 13 C 100.8 55.1 78.9 76.2 72.5 17.1 K 1 H 5.76 3.44 4.46 5.85 13 C 96.9 53.6 62.8 109.1 6116 E. Vinogradov et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Sephadex G50 resulted in the isolation of depolymerized O-chain and three glucans. Lipid A containing compounds were uniformly spread and mixed with other dominating components: [-6)-a- D -Glc-(1-] [-4)-a- D -Glc-(1-] with - 4,6)-a- D -Glc-(1- branching (amylopectin) [-6)-b- D -Glc-(1-] n -3-)-Gro-(1-P-1)-Gro-5 (b-glucan) The linear a-1-6-glucan had highest molecular mass (eluted first from Sephadex G-50 gel permeation column) and was found in minor quantities. Amylopectin was present in the largest amount and constituted  50% of the LPS mass prior to fractional ultracentrifugation, where it can be mostly removed at low speed (27 000 g). The b-glucan 5 had short glucose chains. Its mass spectrum corresponded to 8–15 glucose units with a maximum at 12 units. The same b-glucan was isolated from the precipitate (Ôlipid AÕ), obtained after AcOH hydrolysis of the LPS. Treatment of this precipitate with anhydrous hydrazine led to solubiliza- tion of compound 5, identical to that obtained directly from the LPS after hydrazine treatment. Probably in its native form the b-glucan 5 is acylated with fatty acids and thus is a glycolipid. Its structure was determined by NMR (data will be presented elsewhere). DISCUSSION The LPS of F. tularensis possesses an unusual lipid A structure: its major fraction does not contain phosphate substituents, apparently has reducing glucosamine (residue A) endgroup, and is not acylated at O-3 of GlcN B.Mostof the reported structures of lipid A have phosphate substit- uents at the reducing end [11]. Phosphateless structures with free reducing end glucosamine have been reported in Rhodomicrobium vannielii ATCC 17100 and Rhizobium etli CE3 lipid A [12,13]. R. etli CE3 lipid A contains galact- uronic acid at O-4 of b-GlcN residue; certain other lipids from Aquifex pyrophilus [14] and Caulobacter crescentus (E. Vinogradov & M. B. Perry, unpublished data) contain galacturonic acid both at O-1 of a-GlcN and at O-4 of b-GlcN residues. At the same time F. tularensis LPS preparation contained some of the monophosphorylated form of the lipid A (its backbone was isolated as compound 4), which is not substituted with Kdo. Normally viable bacteria do not produce lipid A unsubstituted with Kdo; the inability to transfer Kdo to the lipid moiety is fatal for the microorganism. However, some bacteria, e.g. Neisseria,can be viable without transfer of Kdo to the lipid moiety [15]. Acyl residues at each acylation position in F. tularensis lipid A can be of two different chain lengths (C14/C16, C16/ C18). Such random distribution of fatty acids between positions of acylation has been found in the lipid A moieties from other bacterial species [16]. The structure of the LPS core part includes Kdo without (or with base labile) substituent at O-4, which has not been observed before. This unusual feauture requires, however, further confirmation since some groups could be lost in harsh conditions of alkaline deacylation. No phosphate substituents in core region have been found. The core part is small and contains mannose in the inner part instead of the more common heptose. Core structures with Kdo substi- tuted by mannose residues were reported in several micro- organisms, including Legionella pneumophila,different Rhizobium species, and some other bacteria [17]. It remains to be determined which of the aforementioned structural features of the lipid A and core of F. tularensis LPS account for the lack of endotoxic and inflammogenic activity of the intact molecule [6]. The presence of longer (compared to the highly endotoxic lipid of E. coli)chain fatty acids, the absence of phosphates and of an acyl group at O-3 of a-GlcN residue could be responsible for the observed weak endotoxic properties of the LPS. Weak endotoxicity might account for the fact that F. tularensis induces relatively little inflammation at sites of infection compared to other facultative intracellular pathogens [18]. Finally, it was noted that the LPS prepared by standard phenol–water extraction was heavily contaminated with an amylopectin-like glucan, a a-(1–6)-linear glucan, and a short chain b-(1–6)-glucan. b-(1–6)-Glucans are uncommon in bacterial sources and to our knowledge only one such glucan has been described in Acinetobacter suis [19]. The presence of glucans can strongly influence the results of biological activity studies with the LPS, at least distorting quantitative results. ACKNOWLEDGEMENTS This work was performed with support from Canadian Bacterial Diseases Network, and by grant AI48474 from the National Institutes of Health, USA. We thank Buko Lindner (Forschungszentrum Borstel, Borstel, FRG) for recording MALDI mass spectra of the lipid A, Bent O. 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Biochem. 269) Ó FEBS 2002 . Structural analysis of Francisella tularensis lipopolysaccharide Evgeny Vinogradov, Malcolm B. Perry and J. Wayne Conlan Institute. 2-N-(3-O- acyl 4 -acyl 2 )-b-GlcN-(1–6)-2-N-acyl 1 )3-O-acyl 3 -GlcN where acyl 1 ,acyl 2 and acyl 3 are 3-hydroxyhexadecanoic or 3-hy- droxyoctadecanoic