Characterization of the lipopolysaccharide and b-glucan of the fish pathogen Francisella victoria William Kay1, Bent O Petersen2, Jens Ø Duus2, Malcolm B Perry3 and Evgeny Vinogradov3 Department of Biochemistry and Microbiology, University of Victoria, BC, Canada Carlsberg Laboratory, Copenhagen, Denmark Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada Keywords core; Francisella; Francisella victoria; lipid A; lipopolysaccharide; O-chain Correspondence E Vinogradov, Institute for Biological Sciences, National Research Council, 100 Sussex Drive, K1A 0R6 Ottawa, ON, Canada Fax: +1 613 9529092 Tel: +1 613 9900832 E-mail: evguenii.vinogradov@nrc.ca (Received 20 January 2006, revised 26 April 2006, accepted May 2006) doi:10.1111/j.1742-4658.2006.05311.x Lipopolysaccharide (LPS) and b-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F victoria as a unique species The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides The nonphosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida The heptoseless core-lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F tularensis and F novicida LPSs, differing in side-chain substituents The linkage region of the O-chain also closely resembled that of other Francisella LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual b-(1–6)-glucan with (glycerol)2phosphate at the reducing end Members of the bacterial genus Francisella belong to the Gram-negative Proteobacteria Their taxonomic position is not completely clear, as no closely related microorganisms have been detected Francisella includes two species: Francisella tularensis and Francisella philomiragia There are four subspecies of F tularensis: tularensis, holarctica, mediasiatica, and novicida Of all subspecies, the F tularensis subspecies tularensis is the most infective and fatal for humans and, due to its very low infective dose, is considered as a biological weapon or bioterrorist agent [1] With the introduction of rapid PCR-based methods of screening of environmental samples, potential new variants of Francisella were detected [2–4] lipopolysaccharide (LPS) of Francisella has unusually low biological activity, and is considered as a potential component of antitularemia vaccines [5–8] Recently a virulent bacterial fish pathogen was isolated from a moribund Tilapia (Oreochromis niloticus niloticus) Tilapia sp are warm water finfish of considerable commercial importance world-wide This pathogen, often and incorrectly referred to as a Rickettsia-like organism, was characterized and identified as a unique Francisella sp by 16S rRNA gene Abbreviations BABA, b-aminobutyric acid; CHA or CHB, cysteine heart agar or broth; Fuc4N, 4-amino-4,6-dideoxygalactose; HPAEC, high-performance anion-exchange chromatography; Kdo, 3-deoxy-D-manno-octulosonic acid; LOS, lipooligosaccharide; LPS, lipopolysaccharide; LVS, live vaccine strain; P, phosphate; QuiN, 2-amino-2,6-dideoxy-D-glucose; Qui3N, 3-amino-3,6-dideoxy-D-glucose; Qui4N, 4-amino-4,6-dideoxy-Dglucose 3002 FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS W Kay et al Francisella victoria lipopolysaccharide sequencing and serological cross-reactivity with other Francisella sp., and it was subsequently named Francisella victoria (unpublished results) Comparative analysis of the LPS from related species is important in order to gain an understanding of the molecular basis of their biological properties, the host specificity and diversity of members of this important bacterial genus, as well as the nature of its pathogenicity The results could also be useful for vaccine development against fish diseases and possibly against tularemia in humans Here we present the results of the structural analysis of the lipopolysaccharide of the first known fish Francisella sp., F victoria Results Silver-stained SDS ⁄ PAGE of F victoria LPS, wholecell proteinase digests or western blot stained with polyclonal antisera to F victoria revealed a predominant immuno-staining band of the large lipooligosaccharide (LOS) component and a smaller less intense band of the core-lipid A component (Fig 1) An additional diffuse band of higher molecular mass components was visible at high sample load Immunostaining using rabbit polyclonal antisera raised to whole cells of High molecular mass components LOS bands Core-Lipid A band Fig Western blot of F victoria LPS products Lane 1: whole F victoria cells treated with Proteinase K overnight at 60 °C Lane 2: Non-precipitated fraction of LPS after overnight ultracentrifugation at 120 000 g Lane 3: LPS ultracentrifuge precipitate F tularensis live vaccine strain (LVS), F novicida or with monoclonal antibodies to LPS from F tularensis or F novicida showed no reaction with the dominant LOS band of F victoria Cross-reactivity was observed with the low molecular weight band of F victoria (data not shown) However, staining of proteinase K-digested samples of F novicida with anti-F victoria antiserum were negative Thus the LPS fractions of whole cells of F victoria were immunochemically distinct from the LPS fractions of the other Francisella sp Monosaccharide analysis (GC MS of alditol acetates) of the whole LPS revealed the presence of rhamnose, fucose, 3-amino-3,6-dideoxyglucosamine (Qui3N), quinovosamine (QuiN), 4-amino-4,6-dideoxyhexosamine (probably a mixture of Fuc4N and Qui4N, as determined from NMR results), mannose, glucose, and glucosamine with dominant peak of glucose 10 times larger than that of any other component High glucose content was due to the presence of glucans in the LPS preparation GC of acetylated or trimethylsilylated (R)-2-butyl glycosides was used to determine the absolute configuration of the monosaccharides, which turned out to be l for Rha and Fuc, and d for QuiN, Qui3N, Glc, Man, and GlcN Configurations of Fuc4N and Qui4N have not been determined because of unclear results LPS was subjected to mild acid hydrolysis, which gave water-insoluble lipid A and water-soluble products Lipid A was purified by conventional silica gel chromatography in a CHCl3–MeOH solvent system Comparison of the 1H-NMR spectra of the unfractionated lipid A and chromatographically fractionated samples indicated that the fraction eluted with 10% MeOH in CHCl3 contained the major component It was used in further studies as ‘lipid A’ Fatty acid analysis of the purified lipid A showed the presence of C14 : 0, C16 : (minor), C16 : (3-OH), and C18 : (3-OH) straight-chain acids Two-dimensional NMR spectra of this product were identical to the spectra of the lipid A from F tularensis [9] The MALDI mass spectrum of the lipid A showed one major peak at m ⁄ z 1391.9, which corresponds to the [M + Na]+ ion of the structure with two GlcN, one C14 : 0, one C16 : (3-OH), and two C18 : (3-OH) fatty acids Smaller peaks at 1364.9 and 1419.9 indicated the presence of the variants with two methylene groups shorter or longer acids, respectively A peak corresponding to the glycosyl cation of unit B was observed at 654.4 Da, corresponding to the acylation of the unit B with C14 : and C18 : (3-OH) acids The same results were observed for F tularensis lipid A [9] For a more detailed analysis of the distribution of O-linked acids, the lipid A was treated with NH4OH FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS 3003 Francisella victoria lipopolysaccharide W Kay et al Fig Structure of the F victoria lipid A and products were analyzed by MALDI mass spectrometry, as described [10] This treatment removes all O-linked fatty acids except those acylating OH-3 of the amide-linked 3-hydroxyacyl groups F victoria lipid A contained only one hydrolyzable acyl substituent at O-3 of residue A Indeed, the mass spectrum of the products showed the new peak of the compound with a mass of 1137.8, corresponding to the loss of C16 : (3-OH) acyl from O-3 of GlcN A residue Together with the above described data, this information can correspond only to the acyl group distribution shown in Fig This experiment also confirmed the previously determined structure of F tularensis and F novicida lipid A Water-soluble products from the mild acid hydrolysis of F victoria LPS were reduced with NaBH4 and separated by size-exclusion chromatography It gave minor amounts of polymeric product, which was shown to be a starch-like glucan, and three oligosaccharide fractions Oligosaccharides were further separated by anionexchange chromatography to give oligosaccharides and (as a mixture), 3, 4, b-glucan (see Scheme 1), and several other products, apparently being fragments of structure Oligosaccharide and b-glucan were additionally purified by high performance anion exchange chromatography (HPAEC) Oligosaccharides 1–4 were analyzed using two-dimensional NMR spectroscopy (COSY, TOCSY, NOESY, heteronuclear single quantum coherence (HSQC), heteronuclear two bond correlation (H2BC), heteronuclear multiple quantum coherence (HMQC)-TOCSY, and heteronuclear multiple bond correlation (HMBC)) and MS Spectra of the simplest oligosaccharide 4, representing the core part of the LPS, were completely assigned in agreement with the proposed structure, consisting of 3004 four mannose residues and one residue of Kdo-ol (Scheme 1, Table 1) ESI MS gave a mass of 888.9 Da, which agreed with the structure Assignment of the spectra of the large oligosaccharides 1–3 presented a significant experimental challenge due to an unusually high number of nonrepeating components and consequential signal overlap even at 800 MHz (Fig 3) Additional problems arose from the presence of the oligosaccharides and as an unseparable mixture, and partial O-acetylation of the Qui3N unit V In order to simplify spectra, oligosaccharides were O-deacetylated and spectra of native and deacylated products were analyzed For the interpretation of the NMR spectra, a new method called H2BC [11–13] was used It produces spectra containing three-bond H–C–C correlations, which makes it possible to identify C-2 signals starting from H-1, and C-5 starting from H-6, as well many other signals NMR analysis of the oligosaccharide showed that it contains all components of the core oligosaccharide Additionally a clearly visible nonreducing end structure was present, consisting of the monosaccharide residues Y-V-U-Z-R, a core-linked sequence L-M-[K-]J-I, and a number of glucose and fucose residues between these two fragments, with an integral intensity of their signals being 1.5–2 times higher than that of above mentioned residues This pointed to the possible presence of loosely defined ‘repeating units’ Relative and anomeric configurations of the monosaccharides were deduced from the proton–proton coupling constants and chemical shifts of proton and carbon signals Connections between monosaccharides were identified on the basis of NOE and HMBC correlations (Table 1) A very large number of the NOE correlations from the H-6 of the 6-deoxysugars was observed, and most of them could be rationalized within the proposed structures (Scheme 1) It should be noted that the signals of the quinovosamine unit I were of low intensity A similar feature was observed previously in the analysis of the repeating unit-core oligosaccharides prepared from F novicida LPS [14], and probably can be explained by the restrained motion of this residue in densely packed structure The ESI MS of the oligosaccharide contained triple and quadruple charged peaks corresponding to a mass of 4381.7 Da, which corresponds to a composition Hex10dHex12HexNAc1dHexNAc3Kdool1 (average mass of 4380.1), thus involving two copies of a fragment P-W-X-[Q-]-T or X-[Q-]-T-[S-]-N (these structures are identical) The spectrum also contained smaller peaks of oligomers of lower and higher mass, differing by hexose units (162 Da) MS-MS analysis generally confirmed structural assignment, but provided no new data FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS W Kay et al Francisella victoria lipopolysaccharide A B C D E F Scheme Structures of the isolated oligosaccharide fragments of the F victoria lipopolysaccharide (LPS) (A) BABA = 3-aminobutyric acid (homoalanine) AcOH hydrolysis products (R1 = Ac or H): full structure = 1; structure ending at PP (PP’) = 2; structure ending at Y (Y’) = 3; structure ending at F (F’) = (B) O,N-deacylated LPS products: full structure = 5; structure ending at PP (PP’ in this case) = 6; structure ending at Y (Y’ in this case) = 7; structure ending at F (F’ in this case) = (C) b-glucan, structures of core-lipid A backbone of different Francisella LPSs: (D) F tularensis, (E) F novicid and, (F) F Victoria regarding the structure of the most obscure region between Fuc R and Fuc L (data not shown) After the determination of the structure of the product 3, the full sequence of the oligosaccharides and was determined by NMR All signals of the components present in the oligosaccharide were found at the same positions except for the substituted Qui3NAc residue Y Oligosaccharide contained three nonsugar components: alanine, 3-aminobutyric acid (homoalanine or BABA), and a novel branched amino acid designated AA HMBC correlations allowed us to trace the BABA-acylated alanine, which was in turn linked to N-4 of terminal Fuc4N residue RR BABA had a free amino group Component AA contained a methyl group and two other protons All proton signals were singlets and showed only NOE correlations between each other Methyl group protons gave HMBC correlations to FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS 3005 Francisella victoria lipopolysaccharide W Kay et al Table NMR data for oligosaccharides 1–8 Components having close chemical shift values are grouped and average data are presented for them Unit, compound Nucleus H⁄C H⁄C H⁄C H⁄C H⁄C H ⁄ C 6a b-Fuc4N RR; b-Fuc4Nac RR; b-Qui4N PP; b-Qui4N PP¢; b-Qui4NAA PP; b-Glc KK; 5,6 b-Glc KK; 1,2 b-Qui3N Y; 5,6 b-Qui3NAc Y; 1,2 b-Qui3N Y¢; b-Qui3NAc Y¢; b-Qui3N V; 5–7 b-Qui3NAc V*; 1–3 deac b-Qui3NAc4Ac V; 1–3 a-Rha U; 1–3, 5–7 a-Rha Z; 1–3, 5–7 a-Fuc R; 1–3, 5–7 a-Glc P; 1–3, 5–7 a-Fuc W; 1–3, 5–7 a-Fuc X; 1–3, 5–7 a-Glc Q; 1–3, 5–7 a-Fuc T; 1–3, 5–7 a-Glc S; 1–3, 5–7 a-Fuc N; 1–3, 5–7 a-Fuc L; 1–3, 5–7 b-Glc M; 5–7 b-Glc M; 1–3 H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C 4.72 103.7 4.54 105.1 4.55 103.5 4.53 103.5 4.47 103.8 4.60 103.5 4.48 103.5 4.82 103.7 4.53 105.1 4.75 104.5 4.53 105.1 4.70 104.7 4.67 105.4 4.72 105.2 5.45 101.7 4.95 97.8 5.01 101.6 5.41 100.8 5.10 101.3 5.15 101.1 5.37 100.8 5.00 101.7 5.39 100.8 5.10 101.3 4.98 100.7 4.61 103.5 4.60 104.2 3.43 71.5 3.46 72.4 3.61 73.8 3.40 73.9 3.58 75.0 3.39 75.4 3.25 74.5 3.67 71.4 3.29 72.7 3.54 72.2 3.25 72.9 3.66 81.0 3.61 79.3 3.73 78.6 4.09 83.4 4.08 71.4 3.92 68.3 3.56 72.8 4.12 75.3 3.85 70.0 3.58 72.8 4.15 70.5 3.56 73.0 4.11 75.5 3.82 69.5 3.39 74.8 3.43 75.5 4.02 70.2 3.85 72.5 3.90 79.3 3.64 72.9 3.79 72.2 3.64 75.4 3.61 75.5 3.36 58.1 3.86 52.2 3.14 58.9 3.82 57.7 3.11 58.9 3.97 57.6 4.16 55.6 3.84 71.6 3.95 79.0 4.01 75.5 3.75 74.0 4.29 70.3 4.11 69.8 3.78 74.2 4.15 76.3 3.73 73.9 4.29 70.3 3.93 70.2 3.52 76.0 3.62 75.5 3.59 56.5 4.19 54.9 3.10 56.5 3.02 57.9 3.74 56.4 3.70 79.5 3.54 80.7 3.66 80.3 3.41 81.6 3.34 72.8 3.15 74.7 3.22 74.3 3.17 74.3 4.63 75.1 3.41 74.3 3.56 73.0 4.03 69.4 3.44 70.6 3.91 82.3 3.91 82.5 3.42 71.0 4.06 80.7 3.44 70.6 3.91 82.3 3.91 82.4 3.56 77.4 3.57 78.0 4.10 68.7 3.81 71.1 3.84 70.2 3.81 70.1 3.74 72.8 3.67 76.0 3.55 76.0 3.82 74.8 3.67 73.9 3.63 75.0 3.56 74.5 3.53 74.3 3.56 74.6 3.78 72.2 3.84 70.1 4.02 70.1 4.53 68.1 3.70 73.8 4.58 69.3 4.42 68.8 3.79 73.9 4.60 69.8 3.71 73.8 4.56 69.3 4.44 68.6 3.63 74.9 3.51 76.7 1.34 16.6 1.16 17.1 1.41 18.0 1.40 18.0 1.30 18.2 3.86 60.9 3.78 61.9 1.46 18.4 1.43 17.9 1.37 17.9 1.36 18.2 1.27 18.0 1.28 18.3 1.19 17.9 1.29 17.8 1.30 17.9 1.19 16.6 3.84 62.0 1.29 16.3 1.46 18.0 3.74 62.1 1.31 17.0 3.85 62.0 1.28 16.4 1.35 17.1 4.02 60.1 4.03 60.7 3006 H ⁄ C ⁄ 6b H ⁄ C 8a ⁄ 8b NOE from H-1 PP3 PP3 KK4 KK4 KK4 4.01 Y4 4.03 Y4 V2 V2 V2 V2 U1,2 U1,2 U1,2 Z3 R3 W4,6 3.71 W1,2 X4,6 T4,6 3.84 T3 N4,6 3.70 N1,2 L4,6 M4,6 3.82 J4,6 3.85 J4,6 FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS W Kay et al Francisella victoria lipopolysaccharide Table (Continued) Unit, compound Nucleus H⁄C H⁄C H⁄C H⁄C a-GlcN K; 5–7 a-GlcNAc K; 1–3 a-Fuc J; 5–7 a-Fuc J; 1–3 b-QuiN I; 5–7 b-QuiNAc I; 1–3 a-Man G; 1–8 b-Man F; 1–3, 5–7 b-Man F¢; 4,8 a-Man H; 1–8 a-Man E; 1–8 Kdo D; 5–8 Kdo C; 5–8 b-GlcN B; 5–8 GlcNol A; 5–8 BABA Ala AA 1,2 H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C H C 5.52 97.2 5.22 99.4 5.10 100.4 4.98 101.2 4.53 101.7 4.56 102.1 5.12 103.0 4.75 101.3 4.72 101.3 5.00 102.2 5.22 102.4 3.32 54.8 3.95 54.8 4.13 69.4 3.93 69.9 2.95 57.5 3.87 56.8 4.09 71.5 4.26 76.2 4.18 77.9 4.06 71.2 4.15 71.2 1.83 102.5 1.97 101.2 3.11 56.4 3.59 56.1 2.34 ⁄ 2.83 38.8 4.26 51.2 4.23 66.5 3.92 70.5 3.80 73.6 4.15 74.0 4.02 73.9 3.48 84.8 3.64 80.9 3.92 71.8 3.86 71.6 3.78 74.9 3.83 72.1 3.95 70.6 2.17 36.0 2.12 36.0 3.61 73.5 3.73 72.4 3.25 53.7 1.40 17.7 3.50 70.3 3.48 71.3 4.20 79.3 4.17 80.3 3.29 74.0 3.32 74.7 3.90 67.3 3.74 78.6 3.64 68.3 3.68 68.1 4.02 77.5 4.01 67.6 4.20 71.3 3.55 70.5 3.93 69.4 1.23 15.1 4.73 100.2 3.65 ⁄ 3.78 63.1 173.8 177.8 170.5 79.0 three carbons, two of them protonated (59.4 and 66.5 p.p.m.) and one quaternary carbon at 79.0 p.p.m Proton signals at 4.23 and 4.73 p.p.m correlated with a quaternary carbon atom and carbonyl carbon atom signals at 176.4 and 170.5 p.p.m These data suggested that AA was a five-carbon dicarboxylic acid with a methyl group at C-3 and amino or hydroxy substituents at positions 2, 3, and For detailed analysis of the structure of AA, LPS was depolymerized with anhydrous HF and Qui4NAA was isolated using reverse-phase HPLC NMR spectra of this monosaccharide (not shown) confirmed its gluco configuration HMBC correlation was observed between C-1 of AA at 170.9 p.p.m and H-4 of the Qui4N, indicating acylation of NH2-4 of Qui4N with one of AA carboxyl groups Spectra contained signals 4.73 59.4 H⁄C 3.96 73.1 3.95 73.4 4.38 68.4 4.41 69.0 3.57 72.9 3.56 73.1 4.02 73.8 3.55 76.5 3.45 78.6 3.55 75.0 4.04 71.9 4.03 68.1 4.27 75.9 3.63 75.3 3.88 71.3 176.4 H ⁄ C 6a 3.89 60.9 3.67 62.1 1.31 16.4 1.28 16.4 1.35 16.5 1.35 18.1 3.63 61.8 3.95 62.0 3.77 62.4 3.79 62.4 3.82 67.6 3.72 73.4 3.66 73.8 3.58 61.8 4.12 71.8 H ⁄ C ⁄ 6b H ⁄ C 8a ⁄ 8b NOE from H-1 3.74 J3 3.78 J3 I3 I3 F4 F4 3.85 F1,2 3.77 E4 3.96 E4 3.91 E6 4.05 C5,7 4.00 71.6 3.78 70.9 3.58 3.81 ⁄ 3.98 64.4 3.63 ⁄ 3.90 65.0 A6 3.74 1.51 23.2 of one N-acetyl group acylating N-4 of the AA The ESI mass spectrum of Qui4NAA showed the molecular mass of 361.3 Da, 18 units less than expected for the linear structure of the AA, which pointed to the lactam formation MS ⁄ MS experiments led to the observation of a signal at m ⁄ z 199.2, corresponding to a cyclic AA component The cyclic structure of the AA was confirmed by the observation of the weak C-5: H-2 HMBC correlation There was no data for the determination of the configuration of chiral atoms Taken together, these experimental data agreed with the structure (Fig 4) As only one Qui4N (residue PP) was present in the oligosaccharides and 2, isolated Qui4NAA represented residue PP monosaccharide Close values of NMR shifts for AA in the oligosaccharides and in the FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS 3007 Francisella victoria lipopolysaccharide W Kay et al Fig Structure of the isolated 4-amino-4,6-dideoxy glucose, N-acylated with the amino acid AA (unit PP-AA in the oligosaccharides) product eluted with the void volume turned out to be starch-like material A second (major) fraction contained large oligosaccharides 5–7 A third fraction contained b-glucan with aglycon, modified due to alkaline conditions; it was not further analyzed The lowest molecular mass component, eluted near the salt peak, contained mostly b-glucan and core oligosaccharide 8, purified further by HPAEC Minor fractions from HPAEC contained the variants of structure with partly degraded lipid A glucosamine due to alkaline conditions of deacylation Products 5–7 were found impossible to separate in conditions used, and they were analyzed in the mixture Oligosaccharide was analyzed by NMR Complete assignment of two-dimensional NMR spectra led to the identification of two a-Kdo residues, one b-GlcN, one glucosaminitol, and four mannose residues The bconfiguration of the Man F followed from the observation of intraresidual strong NOEs between H-1 and H-3, H-5, and also from the low field position of the C-5 signal at 78.6 p.p.m Characteristic NOE between H-3 of the Kdo C and H-6 of the Kdo D residues indicated the attachment of Kdo D in a-configuration to O-4 of Kdo C All glycosidic linkages were identified on the basis of transglycosidic NOE and HMBC [p.p.m.] Fig The 800 MHz 1H-NMR spectrum of the mixture of oligosaccharides and is shown HF-released product indicated that its structure was not modified during HF treatment A number of oligosaccharide fractions not presented in Scheme were isolated after acetic acid hydrolysis, which had no components of the core, and contained mostly fragments of the oligosaccharide chain from unit L to Y or from J to Y, including side chains Their NMR spectra contained many minor signals, mostly of a-glucose As PAGE of the LPS showed the presence of high molecular mass chains, it seems reasonable to believe that these oligosaccharides formed the polymeric chain beyond units Y or RR, and for some reason were cleaved off in both acidic and alkaline conditions Deacylation of the LPS with m KOH in the presence of NaBH4 with subsequent fractionation by gel-chromatography on Sephadex G50 gave four fractions As in the case of acetic acid hydrolysis, the Table NMR data for b-glucan A¢ is nonreducing end residue, A – repeating, A¢ – linked to Gro ⁄ 1¢ Unit b-Glc A¢ b-Glc A b-Glc A¢ Gro B Gro C 3008 H C H C H C H C H C ⁄ 3¢ 6¢ 4.44 103.8 4.45 103.9 4.42 103.5 3.83 ⁄ 3.88 67.3 3.80 ⁄ 3.86 67.3 3.25 74.0 3.26 74.0 3.25 74.0 3.99 70.1 3.83 71.6 3.43 76.6 3.43 76.6 3.34 76.6 3.73 ⁄ 3.87 71.5 3.54 ⁄ 3.61 63.1 3.33 70.6 3.39 70.5 3.38 70.5 3.38 76.9 3.56 75.9 3.56 75.9 3.84 61.7 3.79 69.7 3.77 69.7 3.66 4.15 4.14 FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS W Kay et al Francisella victoria lipopolysaccharide correlations The following NOEs were observed in the product 8: B1A6, C3D6, E1C5, E1C7, F1E4, F1E6, G1F2, G1F3, H1E6, which corresponds to the structure presented on Scheme Analysis of the mixture of oligosaccharides 5–7 by NMR (Fig 5, Table 1) confirmed the sugar backbone structure determined from the analysis of the products 1–3 with all nonsugar components of 1–3 absent in 5–7 Compound had the most complete structure; in the oligosaccharide terminal Fuc4N was missing; in 7, the nonreducing sequence b-Fuc4N-3-b-Qui4N-4b-Glc- was missing (Scheme 1) The structure of b-glucan (Scheme 1) was studied by NMR (Table 2), MS and chemical analysis Monosac- charide analysis showed the presence of glucose and glycerol NMR data indicated that short b-(1–6)-linked glucose oligomers have an aglycon, consisting of two glycerol residues, linked by a phosphodiester bond (31P-NMR signal at 1.08 p.p.m.) Methylation analysis of b-glucan revealed the presence of terminal and 4-substituted glucopyranose Positive-mode MALDI mass spectrum of the b-glucan (Fig 6) contained a series of peaks that could be attributed to ions [M + Na]+ and [M +2Na)1]+, with maximum content of oligomer Glc9, which gave disodium peak at m ⁄ z 1749.2 The same b-glucans were found previously in LPS preparations from F victoria and F tularensis [9,15] 3.0 PP'4 PP4 B2 Y3 I2 K2 U4 P4 Y'3 I4 Y4 Q4 P4 M2 V3,4 RR2 K4 P2 P5 P3 L1:M4 S2 Q2 S3 Y2 W1:P5 Q3 X2 U3+U1:Z3 K3 P1:W2 V5 H3 H1:E6 G3 X4 Y'2 J1:I3 R2 I3 L2 Y'5 T1:W4 L3 Y1:V2 Z3 PP'2 KK2 RR4 M3 F5 B3,5 KK3 KK4 W5:X2 V2 PP'3 PP1:KK4 X5:Q3 PP3 E2 X1:T3 E1:C5 G1:F2 W3 N3 W34 F3 X45 N34 KK6 W45 R35 Z1:R3 I1:F4 RR3 R45 Z1:R4 F1:E4 W23 Z2 T45 X35 N23 T3 T2 V1:U2 T35 X5:T3 J2 J35 J3 M1:J4 J45 F2 N35 J4 T1:N3 W35 E3 W1:X4 R3 R4 X1:T4 Q1:T4 G2 H2 S1:N2 X3 W2 U2 K1:J3 Q1:T3 3.5 M4,5 PP2 4.0 W1:X5 4.5 5.5 5.0 4.5 4.0 Fig Overlap of COSY (green), TOCSY (magenta) and NOESY (black) spectra of the mixture of oligosaccharides 5–7, showing correlations from anomeric protons Intensity is set to high level for clarity, but some important correlations became invisible FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS 3009 Francisella victoria lipopolysaccharide W Kay et al Fig MALDI mass spectrum of the b-glucan, isolated from acetic acid hydrolysis products Discussion The LPS of F victoria contains two variants: a roughtype structure consisting of the core and lipid A, and a much larger structure with a nonrepetitive oligosaccharide linked to the core SDS ⁄ PAGE of the proteinase K-treated whole cells or of the purified LPS appears to reflect this composition with low molecular weight bands, probably representing the lipid A and core oligosaccharide regions, and the more abundant, higher molecular weight bands, probably representing lipooligosaccharide conjugated to the polysaccharide component None of these LPS components were cross-reactive with LPS of the other Francisella sp., with the exception of some nonreciprocal cross-reactivity with F novicida, perhaps due to the similarity of their core-region oligosaccharides These results serve to emphasize the uniqueness of the F victoria oligosaccharide and to confirm it as a unique Francisella sp Lipid A had the same structure as determined earlier for F tularensis [9,16] and F novicida [14,15] LPS with a characteristic nonphosphorylated free reducing end Another variant of the lipid A, which seems to be not substituted with core and has a phosphorylated reducing end, and which has been found in F tularensis and F novicida, was not detected in F victoria The inner core of the LPS of F victoria resembles the core of F tularensis and F novicida in the presence of oligosaccharide fragment b-Man-4-a-Man-5-a-Kdo (marked in bold font together with lipid A backbone, Scheme 1), but it has an additional side-chain Kdo residue and different branching substituents The poly3010 saccharide part is linked to the core via b-N-acetylquinovosamine or its close relative b-N,N-diacetylbacillosamine in all Francisella LPSs (Scheme 1) Oligosaccharide was found to consist of 33 monosaccharide residues and some nonsugar components, which to the best of our knowledge is the largest complex carbohydrate structure elucidated to date The oligosaccharide had no repeating units in the usual sense, although it contained two copies of the same pentasaccharide fragment Its analysis required application of all available NMR methods, and it is still matter of good luck that signals were spread sufficiently to allow interpretation of the spectra Assignment relied strongly on the combined usage of several heteronuclear 1H–13C correlated experiment including the new experiment H2BC, as described recently [13] LPS preparation from F victoria contained two polymers of glucose, a starch-like polymeric material and short b-1–6-glucan, found previously in F tularensis and F novicida [9,15] These components seem to be characteristic for Francisella species Overall the similarity of structural elements of LPS and other components clearly shows that newly discovered F victoria is indeed a new species of Francisella genus There are several unresolved questions concerning Francisella LPS biological activity Thus the role of the ‘starch’-like material and other glucans, coextracted with LPS, is not clear Polymers such as these are conserved microbial structures called ‘pathogen-associated molecular patterns’, which are ligands for pattern recognition receptors expressed FEBS Journal 273 (2006) 3002–3013 ª 2006 The Authors Journal compilation ª 2006 FEBS W Kay et al on various immune cells as part of the innate immunity recognition system [17,18] b-1,3 ⁄ 1,6-Glucans are cell wall components of various bacteria, fungi, and plants, which effect the immune response of various vertebrates species, including fish [19] However, these b-glucan polymers are known to have a schizophrenic activity; at low concentrations they have been shown to be immuno-stimulatory, whereas at higher concentrations they can be immuno-inhibitory [20,21] and seem to modulate the release of potent cytokines induced by LPS Possibly the starch-like polymer and glucans coextracted here with F victoria LPS are immuno-modulatory ancillary polymers A structural understanding of specific polymers, such as those shown here, may shed light on how Francisella sp so effectively evades or suppresses the host immune response and why, after so many years, there are still no efficacious vaccines available Experimental procedures Growth of F victoria F victoria was initially isolated as the predominant Gramnegative pathogen from the kidney of a moribund, apparently wild Tilapia sp., Oreochromis niloticus F victoria grew slowly at