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
1 Department of Biochemistry and Microbiology, University of Victoria, BC, Canada 2 Carlsberg Laboratory, Copenhagen, Denmark 3 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 5 May 2006)
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 eluci- dated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, inclu- ding the first application of H2BC sequence to oligosaccharides. The non- phosphorylated 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 con- tained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side-chain substitu- ents. The linkage region of the O-chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previ- ously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual b-(1–6)-glucan with (glycerol)2phos- phate at the reducing end.
have
samples, potential new variants of environmental Francisella were detected [2–4] lipopolysaccharide (LPS) of Francisella has unusually low biological activ- ity, and is considered as a potential component of antitularemia vaccines [5–8].
commercial
Members of the bacterial genus Francisella belong to the Gram-negative Proteobacteria. Their taxonomic position is not completely clear, as no closely related microorganisms detected. Francisella been includes two species: Francisella tularensis and Franci- sella philomiragia. There are four subspecies of F. tula- rensis: 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 introduct- ion of rapid PCR-based methods of screening of
Recently a virulent bacterial fish pathogen was iso- lated from a moribund Tilapia (Oreochromis niloticus niloticus). Tilapia sp. are warm water finfish of con- siderable importance world-wide. This pathogen, often and incorrectly referred to as a Rick- ettsia-like organism, was characterized and identified as a unique Francisella sp. by 16S rRNA gene
doi:10.1111/j.1742-4658.2006.05311.x
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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-D- glucose.
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-diges- ted samples of F. novicida with anti-F. victoria anti- serum were negative. Thus the LPS fractions of whole cells of F. victoria were immunochemically distinct from the LPS fractions of the other Francisella sp.
sequencing and serological cross-reactivity with other Francisella sp., and it was subsequently named Franci- sella victoria (unpublished results). Comparative analy- sis 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 Francisel- la sp., F. victoria.
fucose,
W. Kay et al. Francisella victoria lipopolysaccharide
Results
Silver-stained SDS ⁄ PAGE of F. victoria LPS, whole- cell proteinase digests or western blot stained with polyclonal antisera to F. victoria revealed a predomin- ant immuno-staining band of the large lipooligosaccha- ride (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
Monosaccharide analysis (GC MS of alditol ace- the whole LPS revealed the presence of tates) of 3-amino-3,6-dideoxyglucosamine rhamnose, (Qui3N), quinovosamine (QuiN), 4-amino-4,6-dide- oxyhexosamine (probably a mixture of Fuc4N and Qui4N, as determined from NMR results), mannose, glucose, and glucosamine with dominant peak of glu- cose (cid:1)10 times larger than that of any other compo- nent. 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 monosac- charides, which turned out to be l for Rha and Fuc, and d for QuiN, Qui3N, Glc, Man, and GlcN. Config- urations of Fuc4N and Qui4N have not been deter- mined because of unclear results.
1
2
3
High molecular mass components
LOS bands
Core-Lipid A band
LPS was subjected to mild acid hydrolysis, which gave water-insoluble lipid A and water-soluble prod- ucts. Lipid A was purified by conventional silica gel chromatography in a CHCl3–MeOH solvent system. Comparison of the 1H-NMR spectra of the unfraction- ated 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 ana- lysis of the purified lipid A showed the presence of C14 : 0, C16 : 0 (minor), C16 : 0 (3-OH), and C18 : 0 (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 : 0 (3-OH), and two C18 : 0 (3-OH) fatty acids. Smaller peaks at 1364.9 and 1419.9 indicated the presence of the variants with two methylene groups shorter or lon- ger 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 : 0 and C18 : 0 (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
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Fig. 1. Western blot of F. victoria LPS products. Lane 1: whole F. victoria cells treated with Proteinase K overnight at 60 (cid:1)C. Lane 2: Non-precipitated fraction of LPS after overnight ultracentrifuga- tion at 120 000 g. Lane 3: LPS ultracentrifuge precipitate.
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.
W. Kay et al. Francisella victoria lipopolysaccharide
Assignment of the spectra of the large oligosaccha- rides 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 1 and 2 as an unsepa- rable mixture, and partial O-acetylation of the Qui3N unit V. In order to simplify spectra, oligosaccharides were O-deacetylated and spectra of native and deacyl- ated 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 iden- tify C-2 signals starting from H-1, and C-5 starting from H-6, as well many other signals.
and products were analyzed by MALDI mass spectr- ometry, 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 substitu- ent 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 : 0 (3-OH) acyl from O-3 of GlcN A residue. Together with the above described data, this informa- tion can correspond only to the acyl group distribution shown in Fig. 2. This experiment also confirmed the previously determined structure of F. tularensis and F. novicida lipid A.
NMR analysis of the oligosaccharide 3 showed that it contains all components of the core oligosaccharide 4. Additionally a clearly visible nonreducing end struc- ture 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 con- stants and chemical shifts of proton and carbon sig- nals. Connections between monosaccharides were identified on the basis of NOE and HMBC correla- tions (Table 1). A very large number of the NOE cor- the 6-deoxysugars was relations from the H-6 of 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.
Water-soluble products from the mild acid hydrolysis of F. victoria LPS were reduced with NaBH4 and separ- ated by size-exclusion chromatography. It gave minor amounts of polymeric product, which was shown to be a starch-like glucan, and three oligosaccharide fractions. separated by anion- Oligosaccharides were further exchange chromatography to give oligosaccharides 1 and 2 (as a mixture), 3, 4, b-glucan (see Scheme 1), and several other products, apparently being fragments of structure 3. Oligosaccharide 4 and b-glucan were addi- tionally purified by high performance anion exchange chromatography (HPAEC).
Oligosaccharides 1–4 were analyzed using two-dimen- sional NMR spectroscopy (COSY, TOCSY, NOESY, heteronuclear single quantum coherence (HSQC), het- eronuclear two bond correlation (H2BC), heteronuclear multiple quantum coherence (HMQC)-TOCSY, and heteronuclear multiple bond correlation (HMBC)) and MS. Spectra of the simplest oligosaccharide 4, represent- ing the core part of the LPS, were completely assigned in agreement with the proposed structure, consisting of
The ESI MS of the oligosaccharide 3 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 con- firmed structural assignment, but provided no new data
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Fig. 2. Structure of the F. victoria lipid A.
W. Kay et al. Francisella victoria lipopolysaccharide
A
B
C
D
E
F
regarding the structure of the most obscure region between Fuc R and Fuc L (data not shown).
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.
After the determination of the structure of the prod- uct 3, the full sequence of the oligosaccharides 1 and 2 was determined by NMR. All signals of the components present in the oligosaccharide 3 were found at the same positions except for the substituted Qui3NAc residue Y. Oligosaccharide 1 contained three nonsugar compo- nents: alanine, 3-aminobutyric acid (homoalanine or
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
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Scheme 1. 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’) = 4. (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) = 8. (C) b-glucan, structures of core-lipid A backbone of different Fran- cisella LPSs: (D) F. tularensis, (E) F. novicid and, (F) F. Victoria.
W. Kay et al. Francisella victoria lipopolysaccharide
Table 1. NMR data for oligosaccharides 1–8. Components having close chemical shift values are grouped and average data are presented for them.
Nucleus NOE from H-1 Unit, compound H ⁄ C 1 H ⁄ C 2 H ⁄ C 3 H ⁄ C 4 H ⁄ C 5 H ⁄ C 6a H ⁄ C 7 ⁄ 6b H ⁄ C 8a ⁄ 8b
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4.72 3.43 4.02 3.59 4.10 1.34 PP3 103.7 71.5 70.2 56.5 68.7 16.6 4.54 3.46 3.85 4.19 3.81 1.16 PP3 105.1 72.4 72.5 54.9 71.1 17.1 4.55 3.61 3.90 3.10 3.84 1.41 KK4 103.5 73.8 79.3 56.5 70.2 18.0 4.53 3.40 3.64 3.02 3.81 1.40 KK4 103.5 73.9 72.9 57.9 70.1 18.0 4.47 3.58 3.79 3.74 3.74 1.30 KK4 103.8 75.0 72.2 56.4 72.8 18.2 4.60 3.39 3.64 3.70 3.67 3.86 4.01 Y4 103.5 75.4 75.4 79.5 76.0 60.9 4.48 3.25 3.61 3.54 3.55 3.78 4.03 Y4 103.5 74.5 75.5 80.7 76.0 61.9 4.82 3.67 3.36 3.66 3.82 1.46 V2 103.7 71.4 58.1 80.3 74.8 18.4 4.53 3.29 3.86 3.41 3.67 1.43 V2 105.1 72.7 52.2 81.6 73.9 17.9 4.75 3.54 3.14 3.34 3.63 1.37 V2 104.5 72.2 58.9 72.8 75.0 17.9 4.53 3.25 3.82 3.15 3.56 1.36 V2 105.1 72.9 57.7 74.7 74.5 18.2 4.70 3.66 3.11 3.22 3.53 1.27 U1,2 104.7 81.0 58.9 74.3 74.3 18.0 4.67 3.61 3.97 3.17 3.56 1.28 U1,2 105.4 79.3 57.6 74.3 74.6 18.3 4.72 3.73 4.16 4.63 3.78 1.19 U1,2 105.2 78.6 55.6 75.1 72.2 17.9 5.45 4.09 3.84 3.41 3.84 1.29 Z3 101.7 83.4 71.6 74.3 70.1 17.8 4.95 4.08 3.95 3.56 4.02 1.30 R3 97.8 71.4 79.0 73.0 70.1 17.9 5.01 3.92 4.01 4.03 4.53 1.19 W4,6 101.6 68.3 75.5 69.4 68.1 16.6 5.41 3.56 3.75 3.44 3.70 3.84 3.71 W1,2 100.8 72.8 74.0 70.6 73.8 62.0 5.10 4.12 4.29 3.91 4.58 1.29 X4,6 101.3 75.3 70.3 82.3 69.3 16.3 5.15 3.85 4.11 3.91 4.42 1.46 T4,6 101.1 70.0 69.8 82.5 68.8 18.0 5.37 3.58 3.78 3.42 3.79 3.74 3.84 T3 100.8 72.8 74.2 71.0 73.9 62.1 5.00 4.15 4.15 4.06 4.60 1.31 N4,6 101.7 70.5 76.3 80.7 69.8 17.0 5.39 3.56 3.73 3.44 3.71 3.85 3.70 N1,2 100.8 73.0 73.9 70.6 73.8 62.0 5.10 4.11 4.29 3.91 4.56 1.28 L4,6 101.3 75.5 70.3 82.3 69.3 16.4 4.98 3.82 3.93 3.91 4.44 1.35 M4,6 100.7 69.5 70.2 82.4 68.6 17.1 4.61 3.39 3.52 3.56 3.63 4.02 3.82 J4,6 103.5 74.8 76.0 77.4 74.9 60.1 4.60 3.43 3.62 3.57 3.51 4.03 3.85 J4,6 b-Fuc4N RR; 5 b-Fuc4Nac RR; 1 b-Qui4N PP; 5 b-Qui4N PP¢; 6 b-Qui4NAA PP; 1 b-Glc KK; 5,6 b-Glc KK; 1,2 b-Qui3N Y; 5,6 b-Qui3NAc Y; 1,2 b-Qui3N Y¢; 7 b-Qui3NAc Y¢; 3 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 104.2 75.5 75.5 78.0 76.7 60.7
W. Kay et al. Francisella victoria lipopolysaccharide
Table 1. (Continued).
Unit, compound Nucleus NOE from H-1 H ⁄ C 1 H ⁄ C 2 H ⁄ C 3 H ⁄ C 4 H ⁄ C 5 H ⁄ C 6a H ⁄ C 7 ⁄ 6b H ⁄ C 8a ⁄ 8b
two of
them protonated (59.4 and three carbons, 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 substitu- ents at positions 2, 3, and 4.
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 lac- tam formation. MS ⁄ MS experiments led to the obser- vation 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).
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
As only one Qui4N (residue PP) was present in the isolated Qui4NAA repre- oligosaccharides 1 and 2, sented residue PP monosaccharide. Close values of NMR shifts for AA in the oligosaccharides and in the
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5.52 3.32 3.74 3.92 3.50 3.96 3.89 J3 97.2 54.8 70.5 70.3 73.1 60.9 5.22 3.95 3.78 3.80 3.48 3.95 3.67 J3 99.4 54.8 73.6 71.3 73.4 62.1 5.10 4.13 4.15 4.20 4.38 1.31 I3 100.4 69.4 74.0 79.3 68.4 16.4 4.98 3.93 4.02 4.17 4.41 1.28 I3 101.2 69.9 73.9 80.3 69.0 16.4 4.53 2.95 3.48 3.29 3.57 1.35 F4 101.7 57.5 84.8 74.0 72.9 16.5 4.56 3.87 3.64 3.32 3.56 1.35 F4 102.1 56.8 80.9 74.7 73.1 18.1 5.12 4.09 3.85 3.92 3.90 4.02 3.63 F1,2 103.0 71.5 71.8 67.3 73.8 61.8 4.75 4.26 3.77 3.86 3.74 3.55 3.95 E4 101.3 76.2 71.6 78.6 76.5 62.0 4.72 4.18 3.96 3.78 3.64 3.45 3.77 E4 101.3 77.9 74.9 68.3 78.6 62.4 5.00 4.06 3.91 3.83 3.68 3.55 3.79 E6 102.2 71.2 72.1 68.1 75.0 62.4 5.22 4.15 4.05 3.95 4.02 4.04 3.82 C5,7 102.4 71.2 70.6 77.5 71.9 67.6 1.83 4.00 2.17 4.01 4.03 3.72 3.81 ⁄ 3.98 102.5 71.6 36.0 67.6 68.1 73.4 64.4 1.97 3.78 2.12 4.20 4.27 3.66 3.63 ⁄ 3.90 101.2 70.9 36.0 71.3 75.9 73.8 65.0 3.11 3.58 3.61 3.55 3.63 3.58 4.73 A6 56.4 73.5 70.5 75.3 61.8 100.2 3.59 3.74 3.73 3.93 3.88 4.12 3.65 ⁄ 3.78 56.1 72.4 69.4 71.3 71.8 63.1 3.25 1.23 2.34 ⁄ 2.83 38.8 53.7 15.1 173.8 4.26 1.40 51.2 17.7 177.8 4.23 4.73 1.51 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 1 Ala 1 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 66.5 79.0 59.4 176.4 23.2 170.5
W. Kay et al. Francisella victoria lipopolysaccharide
(major)
5
4
3
2
[p.p.m.]
Fig. 4. Structure of the isolated 4-amino-4,6-dideoxy glucose, N-ac- ylated with the amino acid AA (unit PP-AA in the oligosaccharides).
HF-released product indicated that its structure was not modified during HF treatment.
product eluted with the void volume turned out to be starch-like material. A second fraction contained large oligosaccharides 5–7. A third fraction contained b-glucan with aglycon, modified due to alka- line 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 8 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.
A number of oligosaccharide fractions not presented in Scheme 1 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 4 m KOH in the pres- ence of NaBH4 with subsequent fractionation by gel-chromatography on Sephadex G50 gave four frac- tions. As in the case of acetic acid hydrolysis, the
Oligosaccharide 8 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 b- configuration of the Man F followed from the observa- tion 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 indi- cated the attachment of Kdo D in a-configuration to O-4 of Kdo C. All glycosidic linkages were identified transglycosidic NOE and HMBC on the basis of
Fig. 3. The 800 MHz 1H-NMR spectrum of the mixture of oligosac- charides 1 and 2 is shown.
Table 2. NMR data for b-glucan. A¢ is nonreducing end residue, A – repeating, A¢ – linked to Gro.
2 4 5 6 Unit 1 ⁄ 1¢ 3 ⁄ 3¢ 6¢
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4.44 3.25 3.43 3.33 3.38 3.84 3.66 b-Glc A¢ 103.8 74.0 76.6 70.6 76.9 61.7 4.45 3.26 3.43 3.39 3.56 3.79 4.15 b-Glc A 103.9 74.0 76.6 70.5 75.9 69.7 4.42 3.25 3.34 3.38 3.56 3.77 4.14 b-Glc A¢ 103.5 74.0 76.6 70.5 75.9 69.7 3.99 Gro B 3.83 ⁄ 3.88 3.73 ⁄ 3.87 67.3 70.1 71.5 3.83 Gro C 3.80 ⁄ 3.86 3.54 ⁄ 3.61 67.3 71.6 63.1 H C H C H C H C H C
correlations. The following NOEs were observed in the product 8: B1A6, C3D6, E1C5, E1C7, F1E4, F1E6, G1F2, G1F3, H1E6, which corresponds to the struc- ture presented on Scheme 1.
that
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 5 had the most complete structure; in the oligosaccharide 6 terminal Fuc4N was missing; in 7, the nonreducing sequence b-Fuc4N-3-b-Qui4N-4- b-Glc- was missing (Scheme 1).
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 ser- could be attributed to ions ies of peaks [M + Na]+ and [M +2Na)1]+, with maximum con- tent 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].
The structure of b-glucan (Scheme 1) was studied by NMR (Table 2), MS and chemical analysis. Monosac-
3.0
PP'4
PP4
B2
Y3
I2
M2
K2
V3,4
PP'2
U4
KK2
P4
Q4 P4
Y'3 I4 Y4
RR2
3.5
K4
M4,5
L1:M4
V5
P2
RR4
PP2
Y2
S2 Q2
M3
PP'3
Y'2
F5 B3,5
W1:P5
J1:I3
PP1:KK4
I3
S3
P5 P3
X5:Q3
Q3
Y'5
L2
H3 H1:E6
KK3 KK4 W5:X2
X2
G3
V2
PP3
W34
Y1:V2
U3+U1:Z3
X4
R2
T1:W4 L3 Z3
F3
W45
N34
X45
K3
KK6
E3
W1:X4
I1:F4
4.0
RR3
R3 R4
R35 R45
X1:T4
Q1:T4
G2
F1:E4
W23
P1:W2
H2
Z1:R3 Z1:R4 Z2
T45
X3
X35
N23
S1:N2
W2
U2
E2
T3 T2
V1:U2
T35
X5:T3
K1:J3 Q1:T3
J35
X1:T3
M1:J4
J45
F2
N35
J2 J3 J4
N3
E1:C5
W3
T1:N3
W35
G1:F2
W1:X5
4.5
5.5
5.0
4.5
4.0
W. Kay et al. Francisella victoria lipopolysaccharide
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Fig. 5. 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.
W. Kay et al. Francisella victoria lipopolysaccharide
Fig. 6. MALDI mass spectrum of the b-glucan, isolated from acetic acid hydrolysis products.
Discussion
close
its
saccharide part is linked to the core via b-N-acetylqui- relative b-N,N-diacetyl- novosamine or bacillosamine in all Francisella LPSs (Scheme 1).
Oligosaccharide 1 was
same pentasaccharide
fragment.
Its
several heteronuclear
found to consist of 33 monosaccharide residues and some nonsugar compo- nents, which to the best of our knowledge is the lar- gest complex carbohydrate structure elucidated to date. The oligosaccharide had no repeating units in the usual sense, although it contained two copies of the 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 spec- relied strongly on the combined tra. Assignment 1H–13C correlated usage of experiment including the new experiment H2BC, as described recently [13].
The LPS of F. victoria contains two variants: a rough- type structure consisting of the core and lipid A, and a much larger structure with a nonrepetitive oligosaccha- ride 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 these LPS components were component. None of cross-reactive with LPS of the other Francisella sp., with the exception of some nonreciprocal cross-reactiv- ity with F. novicida, perhaps due to the similarity of their core-region oligosaccharides. These results serve to emphasize the uniqueness of the F. victoria oligosac- charide and to confirm it as a unique Francisella sp.
LPS preparation from F. victoria contained two pol- ymers 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 simi- larity of structural elements of LPS and other compo- nents clearly shows that newly discovered F. victoria is indeed a new species of Francisella genus.
fragment
conserved microbial
structures
There are several unresolved questions concerning Francisella LPS biological activity. Thus the role of the ‘starch’-like material and other glucans, coex- is not clear. Polymers such as tracted with LPS, called are these ‘pathogen-associated molecular patterns’, which are ligands for pattern recognition receptors expressed
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 redu- cing 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 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 poly-
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resuspended
once with NaCl ⁄ Pi,
species,
low concentrations
suppresses
on various immune cells as part of the innate immu- nity 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 including fish [19]. However, vertebrates these b-glucan polymers are known to have a schizo- phrenic activity; at 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 the host sp. so effectively evades or so many years, immune response and why, after there are still no efficacious vaccines available.
whole cells were grown in CHB at 28 (cid:1)C overnight, harves- ted, washed in SDS ⁄ PAGE sample buffer and 50 lL samples digested with 5 lL Proteinase K (1 mgÆmL)1) for 2 h at 60 (cid:1)C and boiled for 10 min to stop the digestion. For western blotting, the prospective antigens were electrophoretically transferred to nitrocellulose membranes for 1 h at 50 mAÆgel)1 (Bio-Rad, Hercules, CA, USA). These transblots were blocked using 5% w ⁄ v skimmed milk ⁄ NaCl ⁄ Pi ⁄ Tween-20 and reacted for 1 h at room temperature with a 1 : 3000 dilution of rabbit polyclonal antisera (anti-F. tularensis LVS; anti-F. novicida; anti-F. victoria) or mouse mAb (anti-F. tularensis LVS LPS; in NaCl ⁄ Pi)0.5% Tween-20. The anti-F. novicida LPS) membranes were then washed and further reacted with a 1 : 4000 dilution of second antibody, goat antirabbit IgG conjugated to alkaline phosphatase (Caltag Laboratories, Burlingame, CA, USA) and developed for 4 h at room tem- perature with 5-bromo-4-chloro-3-indolyl phosphate and 4- nitro blue tetrazolium chloride. Francisella sp. and LPS-spe- cific antisera were kindly provided by F. Nano, University of Victoria, BC, Canada.
W. Kay et al. Francisella victoria lipopolysaccharide
Experimental procedures
Growth of F. victoria
F. victoria was initially isolated as the predominant Gram- negative pathogen from the kidney of a moribund, appar- ently wild Tilapia sp., Oreochromis niloticus. F. victoria grew slowly at <30 (cid:1)C on cysteine heart agar or broth (CHA or CHB).
For large-scale LPS preparations F. victoria cells (300 g wet mass) were extracted by stirring with 50% aqueous phenol (500 mL, 70 (cid:1)C, 15 min). The cooled extract was diluted by equal amounts of water, dialyzed against tap water until phenol-free and lyophilized. The respective resi- dues were resuspended in 50 mL 0.02 m sodium acetate, pH 7.0, and treated sequentially with RNase, DNase and proteinase K (37 (cid:1)C, 2 h each). Enzyme-treated samples were subjected to ultracentrifugation (120 000 g, 12 h, 4 (cid:1)C) and the precipitated gels were dissolved in water and lyophilized to yield 350 mg of LPS.
Mild acid hydrolysis
Stock cultures were held at )80 (cid:1)C in 25% glycerol and cultured at 28 (cid:1)C on CHA. Three colonies were inoculated into 0.2 mL of CHB, grown at 28 (cid:1)C and sequentially scaled up to 5 mL and 100 mL of CHB, which was then used to inoculate a 25-L CHB fermentation broth. Fermen- tation was carried out in CHB at 28 (cid:1)C in a Chemap fer- mentor fitted with Chemap’s Fundafom foam breaker and a bottom-driven, three-tier, flat-bladed impeller system. Aeration was initially adjusted to 25 LÆmin)1. The control systems provide proportional control of impeller speed, air- flow, and temperature; pH and aeration were under control. Growth was monitored at A600 until the culture grew to (cid:1)4 A600 (48 h). Data from the fermentor was logged using a PC and GenesisTM process control software. pH was con- trolled by the addition of 5% H2SO4. For harvest and con- centration a high capacity Millipore PelliconTM tangential flow filtration system was used and cells were finally pellet- ed by centrifugation at 10 000 g.
PAGE of F. victoria LPS
Aqueous phase LPS (100 mg) was hydrolyzed with 2% acetic acid (6 mL) at 100 (cid:1)C for 4 h and, following removal of precipitated lipid A by centrifugation at 30 000 g, the concentrated water soluble products were fractionated by Sephadex G-50 chromatography to yield for fractions I–IV. Fractions II–IV were further separated by anion exchange chromatography on Hitrap Q column (5 mL, Amersham, Piscataway, NJ, USA) in a 3 mLÆmin)1. gradient of water (first 20 min) to 1 m NaCl over 1 h with UV detection at 220 nm and sugar detection by charring aliquots from each fraction on silica gel TLC plates after dipping in 2% H2SO4 in MeOH. Thus acidic oligosaccharides 1–3 were isolated from fraction II, b-glucan from fraction III, and oligosac- charide 4 from fraction IV. Neutral oligosaccharides eluted with water were not numbered.
O,N-Deacylation of the LPS
LPS samples (1 mgÆmL)1) were boiled in SDS sample buf- fer for 10 min and 50 lL used for SDS ⁄ PAGE according to the Laemmli method as modified. SDS gels were washed for 15 min in dH2O and chemically stained for LPS with Gelcode (Pierce, Rockford, IL, USA). For western blotting,
LPS (80 mg) was dissolved in 4 m KOH (4 mL) containing NaBH4 (50 mg), kept overnight at 100 (cid:1)C, and neutralized
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to Francisella sp. and to J. Burian and J. Barlow (Mic- rotek Intl. Ltd, Saanichton, BC, Canada) for the fer- mentor growth of F. victoria.
W. Kay et al. Francisella victoria lipopolysaccharide
References
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