Báo cáo khoa học: Structural characterization of the lipopolysaccharide O-polysaccharide antigen produced by Flavobacterium columnare ATCC 43622 potx

Structural characterization of the lipopolysaccharide O -polysaccharide antigen produced by Flavobacterium columnare ATCC 43622 Leann L. MacLean 1 , Malcolm B. Perry 1 , Elizabeth M. Crump 2 and William W. Kay 2 1 Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada; 2 Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada The structure of the antigenic O-chain polysaccharide of Flavobacterium columnare ATCC 43622, a Gram-negative bacterium that causes columnaris disease in warm water fish, was determined by high-field 1D and 2D NMR techniques, MS, and chemical analyses. The O-chain was shown to be an unbranched linear polymer of a trisaccharide repeat- ing unit composed of 2-acetamido-2-deoxy- D -glucuronic acid ( D -GlcNAcA), 2-acetamidino-2,6-dideoxy- L -galactose ( L -FucNAm) and 2-acetamido-2,6-dideoxy- D -xylo-hexos- 4-ulose ( D -Sug) (1 : 1 : 1), having the structure: Keywords: Flavobacterium columnare; lipopolysaccharide; NMR. Flavobacterium columnare, formerly referred to as Flexi- bacter columnaris or Cytophaga columnaris [1], is a Gram- negative bacterium which causes columnaris disease [2] in warm water fish, a disease that is the second leading cause of mortality in pond raised catfish in the south-eastern United States. The virulence factors of F. columnare are relatively unknown, but it has been suggested that, in pathogenesis, adhesion of the bacterium may be related to its surface polysaccharide constituents [3–6]. This investigation was directed towards characterization of the lipopolysaccharide (LPS) and putative capsule produced by the bacterium, as a first step in identifying their possible role in pathogenesis in fish. In addition, it was considered that characterization of the LPS O-polysaccharide (O-PS) antigen would provide a structural knowledge basis for the development of a specific antibody diagnostic agent and possible target molecules for a conjugate based vaccine. Experimental procedures Bacterial culture F. columnare (ATCC 43622, NRCC 6160) was grown at 16 °C in a 52-L fermentor in medium of composition: tryptone, 4 g; yeast extract, 0.4 g; MgSO 4, 0.5 g; CaCl 2 , 0.5 g; sodium acetate, 0.2 g; maltose, 10 gÆL )1 ;pHwas adjusted to 7.00 with 0.1 M NaOH. A 2.5-L inoculum grown at 22 °C was used, with stirring at 200 r.p.m. and dissolved oxygen at 20%. Cells were killed with 1% phenol (final concentration, 2 h at 4 °C) in late exponential phase at 25.5hgrowth(A 600 ¼ 3.34). After acidification with acetic acid to pH 4 at 0 °C to break the gel-like constitution, the suspended cells were harvested by centrifugation (yield  300 g wet paste). Preparation of LPS and O -PS F. columnare cells (300 g wet paste) were extracted for 15 min at 65 °C with vigorously stirred 50% (w/v) aqueous phenol (1.2 L), and, after cooling (4 °C) and low-speed centrifugation, the separated water and phenol layers were collected by aspiration, and dialyzed against running water until free from phenol. The lyophilized dialyzed retentates were dissolved in sodium acetate (0.02 M , pH 7.0, 80 mL) and then treated sequentially with RNase, DNase and proteinase K (37 °C, 2 h each). The digests were cleared by low-speed centrifugation (4000 g) and then subjected to ultracentrifugation (105 000 g,12h,4°C). Only the phenol phase-soluble product afforded a precipitated LPS gel. The gel was ½!4Þ-b-d-GlcpNAcA-ð1!4Þ-a-l-FucpNAm-ð1!3Þ-a-d-Sugp-ð1! 33 "" Ac Ac Correspondence to M. B. Perry, Institute for Biological Sciences, National Research Council, Ottawa, Canada K1A 0R6. Fax: + 1 613 941 1327, Tel.: + 1 613 990 0837. Abbreviations: D -Sug, 2-acetamido-2,6-dideoxy- D -xylo-hexos-4-ulose; D -GlcNAcA, 2-acetamido-2-deoxy- D -glucuronic acid; L -FucNAm, 2-acetamidino-2,6-dideoxy- L -galactose (2-acetamidino-2-deoxy- L -fucose); LPS, lipopolysaccharide; O-PS, O-polysaccharide; CPS, capsular polysaccharide. (Received 21 May 2003, revised 25 June 2003, accepted 27 June 2003) Eur. J. Biochem. 270, 3440–3446 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03736.x dissolved in water and lyophilized to yield LPS (1.68 g), which was used in all further studies. The addition of acetone (6 vol.) to the supernatant from the above ultracentrifugate remaining after the collection of LPS afforded a precipitate (94 mg), which, on Sephadex G- 50 column chromatography, yielded a void-volume elution product (55 mg) tentatively identified as capsular polysac- charide (CPS). O -PS LPS (1.40 g) was delipidated by treatment with 1% (v/v) acetic acid (100 mL) at 100 °C for 2 h and, after removal of precipitated lipid A (170 mg), the lyophilized water-soluble products were fractionated by Sephadex G-50 column chromatography to yield an O-PS fraction (K av 0.03–0.12, 390 mg) and a low-molecular-mass putative core oligosac- charide fraction (K av 0.75, 110 mg). Chromatography and electrophoresis Descending preparative paper chromatography was per- formed on water-washed Whatman 3 MM paper using butanol/ethanol/water (4 : 1 : 5, by vol., top layer). Detec- tion was with 2% ninhydrin in acetone, and mobilities are quoted relative to D -glucosamine/HCl (R GN ). GLC was performed with an Agilent 6850 Series gas chromatograph fitted with a flame ionization detector and a Phenomenex Zebron capillary column ZB-50 (30 m · 0.25 mm · 25 lm) using a temperature program 170 °C (delay 4 min) to 240 °Cat4°CÆmin )1 . GLC/MS was performed under the same conditions using a Hewlett–Packard 5985 GLC/MS system and an ionization potential of 70 eV. Retention times are quoted relative to hexa-O-acetyl- D -glucitol (T G ¼ 1.00). Polysaccharide was separated by Sephadex G-50 column (2 · 85 cm) chromatography using 0.05 M pyridinium acetate (pH 4.5) as the mobile phase, and the eluate was continuously monitored using a Waters R403 differential refractometer. LPS samples (2 lg) were electrophoresed in 14% poly- acrylamide in the presence of deoxycholate. Bands were detected using the silver-staining directions of Tsai & Frasch [7]. NMR spectroscopy 1 Hand 13 C NMR spectra were recorded on a Varian Inova 400 spectrometer with samples in 99% D 2 Oat 55 °C, and internal acetone standard (2.225 p.p.m. for 1 H and 31.07 p.p.m. for 13 C) employing standard COSY, TOCSY (mixing time 80 ms), NOESY (mixing time 200 ms), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple-bond correlation (gHMBC) (optimized for 5 Hz long-range coupling constant). Chemical procedures Quantitative conversion in the O-PS of the acetamidino function into an acetamido function was effected by treatment of the O-PS with 5% aqueous triethylamine (3 h, 70 °C) as previously described [8] to yield the modified O-PS. Simultaneous reduction of the carboxy function of the uronic residue C and the 4-keto function of residue B was made by treatment of the native O-PS(47mg)inwater (10 mL) with 1-(3-dimethylaminopropyl)-3-ethyl carbodi- imide (150 mg) maintained at pH 4.7 over 4 h followed by reduction at 0 °C by sodium borodeuteride (100 mg, 2 h), followed by neutralization with acetic acid, dialysis against distilled water, and recovery of the reduced O-PS (40 mg) in the void-volume fraction from Sephadex G-50 gel-filtration chromatography. A similar preparation of reduced O-PS was made by reduction with NaBH 4 under the same experimental conditions. General methods Hydrolyses were carried out in sealed tubes with either 6 M HCl (100 °C, 3 h) or 2 M trifluoroacetic acid (105 °C, 4 h), and samples were concentrated to dryness in a stream of nitrogen and examined directly or after derivatization. Alditol acetates were prepared after reduction (NaBD 4 or NaBH 4 ) and acetylation (Ac 2 O) of isolated aldoses, as previously described [8]. The absolute configuration of derived 2-acetamido-2-deoxyhexoses was confirmed by GLC analysis of their acetylated 2-(S)-butyl glycosides, prepared under previously described conditions [9]. Optical rotations were measured at 20 °C in 10-cm microtubes, using a Perkin-Elmer 243 polarimeter. Results Fermenter-grown cells of F. columnare were extracted by a modified hot phenol/water method [10], and a S-type LPS, found almost exclusively in the phenol phase of the cooled extract, was obtained in 12% yield by ultracentrifugation of the concentrated dialyzed extract. Deoxycholate/PAGE analysis of the LPS gave a typical ladder-like banding pattern in which the step separations suggested that the LPS was composed of repeating trisaccharide units [11]. On treatment with 6 vol. acetone, the ultracentrifugate afforded a precipitate which, on Sephadex G-50 gel filtration, gave a void-volume fraction ( 2% yield) of a glycan tentatively identified as CPS. A lower-molecular-mass fraction (K av 0.7, 180 mg) which gave a strong colorimetric (phenol/H 2 SO 4 ) reaction for carbohydrate contained glycopeptides in which the oligosaccharide moieties had similar structure and composition (results not reported) to those previously found in glycoproteins produced by Flavobacterium meningosepticum [12]. The LPS was delipidated by treatment with hot dilute acetic acid and after removal of precipitated lipid A (8%), the O-PS (86%) was collected in the void-volume fraction obtained by Sephadex G-50 gel filtration of the water- soluble products. The O-PS had [a] D )90.1 ° (c 8.9, water) Anal. C, 44.61; H, 6.18; N, 7.12% and ash, nil. GLC analysis of the acetylated 2 M trifluoroacetic acid (105 °C, 4 h) O-PS hydrolysis products gave a low yield ( 2%) mixture of mannose, galactose and L -glycero- D -manno-heptose. These glycoses probably originate from a core oligosaccharide component; however, no significant hydrolysis products from the major O-PS component were detected. Ó FEBS 2003 Flavobacterium columnare polysaccharide (Eur. J. Biochem. 270) 3441 The 1D 1 H-NMR spectrum of the O-PS showed inter alia: three anomeric glycose H1 proton signals at 5.14 (J 1,2 2.2 Hz), 4.97 (J 1,2  3Hz)and4.70(J 1,2 8.8 Hz) p.p.m. with J 1,2 couplings indicative of two a-linkage and one b-linkage, respectively; two methyl signals at 1.21 and 1.17 p.p.m. (6H) characteristic of two 6-deoxyhexose residues; an N-acyl substituent at 2.25 p.p.m. (3H); and four signals (2.10–1.93 p.p.m.) characteristic of methyl signals of two N-acetyl and two O-acetyl substituents. The 13 C-NMR spectrum of the O-PS (Fig. 1) showed inter alia three anomeric signals at 102.6 (J C-1,H-1 164 Hz), 97.1 (J C-1,H-1 172 Hz) and 97.0 (J C-1,H-1 180 Hz) p.p.m. having J C-1,H-1 coupling constants indicative of one b-link- age and two a-linkages, respectively, together with a sharp singlet at 93.9 p.p.m. subsequently identified as the C4 resonance of a 4-ketohexose residue. Also present were two sharp singlets at 15.8 and 11.9 p.p.m. characteristic of methyl shifts of 6-deoxyhexose residues, signals at 167.0 p.p.m. (C¼N) and 19.8 p.p.m. (CH 3 -C¼N) charac- teristic of acetamidino groups, and ring carbon signals at 55.3, 52.8 and 51.1 p.p.m. indicative of C-N-linked sub- stituents, together with a total of four signals subsequently assigned to methyl groups of two N-acetyl substituents (22.8 and 23.0 p.p.m.), and two O-acetyl substituents (21.1 and 20.9 p.p.m.). Five signals attributed to carbonyl substituents were observed in the 175.7–173.6 p.p.m. region. The preliminary data suggest that the O-PS is a polymer of regular trisaccharide repeating units composed of three aminoglycose residues. The chemical shift assignments in the 1 H-NMR and 13 C-NMR spectra and the characterization of the glycose components in the O-PS were determined from the appli- cation of COSY, TOCSY, NOESY and 1 H, 13 C-HSQC and HMBC experiments (Table 1, Fig. 2). For the analysis, Fig. 1. 13 C-NMR spectrum of F. columnare O-PS recorded at 55 °C (125 MHz). Table 1. 1 H and 13 C NMR chemical shifts of the native LPS O-PS from F. columnare ATCC 43622. Spectra run in D 2 Oat55°C with internal acetone reference (2.225 p.p.m. for 1 H and 31.07 p.p.m. for 13 C). Coupling constants (Hz) are given in parentheses. Tentative assignments for residue A: N-H7 (8.83 p.p.m.) and N-H7 1 (8.57 p.p.m.) at 35 °C(10%D 2 O/90% H 2 O, v/v). Glycose residue Chemical shift (p.p.m.) H1/C1 H2/C2 H3/C3 H4/C4 H5/C5 H6/C6 A 5.14 (2.2) 4.28 (10.2) 5.16 (nr) 4.13 (2) 4.57 1.17 97.1 (172) 51.1 71.5 78.8 67.9 15.8 B 4.97 (3) 4.24 (3.2) 3.78 (nr) – 3.91 1.21 97.0 (180) 52.7 77.7 93.9 70.1 11.9 C 4.70 (8.8) 3.93 (10.0) 5.26 (9.8) 4.08 (10.0) 3.79 – 102.6 (164) 55.4 76.5 74.1 77.8 175.4 3442 L. L. MacLean et al.(Eur. J. Biochem. 270) Ó FEBS 2003 glycose residues were arbitrarily labeled A–C in order of decreasing chemical shifts of the anomeric protons. Glycose A was initially identified as a-FucpNAm. In a COSY experiment, overlapping correlation peaks of H1A to H2A and H2A to H3A were observed due to O-acetylation at O3A causing an upfield shift of its signal to 5.16 p.p.m. A correlation cross-peak for H4A to H5A was not observed in the COSY spectrum because of the small scalar coupling (H 4,5  2 Hz), but was evident from TOCSY data linking H4A to the H6A methyl signal at 1.20 p.p.m. (3H). A direct correlation of H2A to C2A (51.1 p.p.m.) in HSQC and long-range HMBC and correlations from the N-acyl proton (2.25 p.p.m.) to the carbonyl signal at 167.0 p.p.m. were characteristic of an acetamidino group, thus identifying A as an a-FucpNAm residue, the a-configuration being assigned from consid- eration of the anomeric proton and carbon coupling constant data (J Hl,2  2Hz,J C-1,H-1 172 Hz). From NMR data, residue C was assigned the b-gluco- pyranose configuration from its observed large ring region J H,H coupling constants for J 2,3 , J 3,4 and J 4,5 ( 10 Hz), and from its anomeric coupling constants, J C-1,H-1 164 Hz, and J 1,2 8.8 Hz. Thecorrelation of H2C tothe C2C at55.4 p.p.m. is consistent with the presence of a C2 acetamido substituent, and the lack of a proton at C6, considered in conjunction with the long-range correlation of H5C to the carbonyl shift at 175.4 p.p.m., seen in a HMBC experiment, is consistent with the presence of a C6 carboxylic acid function and allows C to be identified as a b-GlcpANAc residue. Residue B was identified as an a-linked 2-acetamido-2,6- dideoxyhexos-4-ulose residue from further NMR data. The observed correlation from H2B to the corresponding C2B in an HSQC experiment, the anomeric coupling constants J 1,2 of 3 Hz and J C-1,H-1 of 180 Hz, considered in conjunction with the fact that connectivities could only be followed from H1B to H2B and H3B, and from the methyl resonance of H6B to H5B with no evidence of connectivities to any proton signals at C4B. The presence of a C4 keto group function and the absence of a proton at C4B was further supported from an observed long-range correlation between H3B and the C4B carbon signal at 93.8 p.p.m. seen in an HMBC experiment, thus identifying B as an a-linked 2-acetamido-2,6-dideoxy-xylo-hexos-4-ulose residue. Further characterizations of the O-PS component glycoses were made from chemical studies. Residue A was identified as 2-acetamidino-2,6-dideoxy- L -galactose after its conversion in the O-PS into its corresponding 2-acetamido derivative by treatment with hot aqueous trimethylamine. The quantitative transformation was verified from the 1D 1 H-NMR spectrum of the modified polymer in which a shift of the characteristic carboxy resonance at 167 (Am) in the native O-PS to 175.3 (Ac) p.p.m. in the modified O-PS was observed. The HCl hydrolysate of the modified O-PS, in contrast with the native O-PS, gave a single aminoglycose product, which was isolated by preparative paper chroma- tography and identified as 2-amino-2,6-dideoxy- L -galactose HCl (R GN 1.47) from its specific optical rotation {[a] D )81 ° (c0.2,water).Lit.[a] D –95° [13]}, the identity of its 1 H- NMR spectrum with that of an authentic sample, and the fact that its reduced (NaBD 4 ) and acetylated product on GLC/MS gave a single peak corresponding in retention time (T G 0.93) and mass spectrum to an authentic sample of 1,3,4,5,-tetra-O-acetyl-2-acetamido-2,6-dideoxy- D -galact- itol-[1- 2 H]. The hexuronic acid component C was identified as 2-acetamido-2-deoxy- D -glucuronic acid from the isolation Fig. 2. 1 H- 13 CHSQCshiftcorrelationmap of the spectral regions 1 H (1.0–5.5 p.p.m.) and 13 C (10–104 p.p.m.) of the F. columnare O-PS with resonances labeled for residues A, B and C. Ó FEBS 2003 Flavobacterium columnare polysaccharide (Eur. J. Biochem. 270) 3443 of 2-amino-2-deoxy- D -glucose-[6- 2 H 2 ], from the hydrolysis product of the reduced (NaBD 4 ) carbodiimide-activated O-PS. The latter glycose isolated by preparative paper chromatography (R GN 1.00) was identified by GLC/MS of its reduced (NaBD 4 ) acetylated derivative 1,3,4,5,6- penta-O-acetyl-2-acetamido-2-deoxyglucitol-[1- 2 H, 6- 2 H 2 ] (T G 1.22), and its D -configuration was confirmed from the specific optical rotation of its hydrochloride derivative {[a] D +67° (c0.3,water).Lit.[a] D +72°} and by GLC analysis of its derived acetylated 2-(S)-butyl glycosides [11]. Residue B was identified as 2-acetamido-2,6-dideoxy- D - xylo-hexos-4-ulose (D-Sug). The above preparative paper chromatography of the hydrolysed reduced (NaBD 4 ) carbodiimide-activated O-PS also yielded two separated aminoglycose fractions identified as a mixture of 2-amino- 2,6-dideoxy- D -(and L )galactose {R GN 1.48; [a] D )4 ° (c 0.2, water) [13]} and 2-amino-2,6-dideoxy- D -glucose {R GN 1.83; [a] D +52° (c 0.4, water); Lit. [a] D +50° [14]}, in approximately equal yield. GLC/MS of their individual reduced (NaBH 4 ) and acetylated alditiol derivatives gave single peaks corresponding in retention times to 1,3,4,5- tetra-O-acetyl-2-acetamido-2,6-dideoxygalactitol (T G 0.93) and 1,3,4,5-tetra-O-acetyl-2-acteamido-2,6-dideoxyglucitol (T G 0.90) standards. The mass spectrum of each derivative showed a fragmentation pattern with characteristic ions of the C1–C2 fragment at m/z 144, 102, 84, and 60 showing that C1 was not deuterium labeled. However, the expected M+1) 60 ¼ 317 molecular-ion and the expected frag- ment ions at m/z 261 (C2 to C6, 303–42, loss of ketene) confirmed that deuterium labeling was present. The chro- matographically isolated 2-amino-2,6-dideoxygalactose fraction was a mixture of the D -and L -forms of the aminoglycose, as evidenced from its optical rotation, and from GLC analysis of its acetylated 2-(S)-butyl glycoside derivatives. This finding is consistent with this fraction being composed of a L -FucN component originating from the O-PS residue A and the D -FucN from the reduced residue B. The isolation of optically pure 2-amino-2,6-dideoxy- D - glucose (D-QuiN), the major reduction product of residue B, further confirms the D -configuration assigned to residue B. Preparative paper chromatographic separation of the hydrolysis products of NaBH 4 -reduced carbodiimide- activated O-PS afforded the hydrochloride derivatives of 2-amino-2-deoxyglucose, 2-amino-2,6-dideoxyglucose, and 2-amino-2,6-dideoxygalactose, the 1 H-NMR spectra of which were identical with those of authentic reference glycoses, and further confirms their characterization. The combined MS data and the isolation of the two aminoglyc- oses with the respective D -galacto and D -gluco configura- tions (epimers at C4) establishes that B is a 4-ketohexose (or 4-acetal derivative) and, from its anomeric proton and carbon chemical shift and coupling constant data, is present in the a- D -hexopyranosyl configuration in the O-PS, and is a 2-acetamido-2,6-dideoxy-a- D -xylo-hexos-4-ulose residue. The sequence of the glycose residues and their linkage positions in the O-PSwereestablishedfrom1Dand2D NOE and long-range multiple-bond 1 H- 13 C(HMBC) correlations experiments. Interresidue NOEs were seen from H1B to H4C andtoitsownH2B,fromH1A to H2A and across the glycosidic bond to H3B,andalsofrom H1C to H3C and H5C and across the ring to H4A.HMBC experiment results were consistent with the proton NMR data showing correlations between C1B (97.0 p.p.m.) to H4C,fromC1C (102.6 p.p.m.) to H4A and from C1A (97.1 p.p.m.) to H3B, thus defining the sequence and linkage position in the O-PS repeating trisaccharide units as fi4)-b-C-(1fi4)-a-A-(1fi3)-a-B-(1fi, leading to a basic repeating unit with the structure: Consistent with the above conclusion, the NMR analysis of the native O-PS showed that the chemical shifts of the linkage position carbon atoms C4A,C3B,andC4C experience significant deshielding, further confirming the linkage position assignments. As NMR data indicated the presence of two O-acetyl substituents in the native O-PS, they can only be located at the available O3 positions of residues A and C. Partial de-O-acetylation of the O-PS with dilute ammonium hydroxide (50 °C,1h)resultedinthe hydrolytic removal of the acetyl substituent on residue A (a- L-FucpNAm) and partial ( 20%) removal from residue C (b-D-GlcpNAcA). The de-O-acetylation of A effected deshielding of C3A (71.5–68.2 p.p.m.) and H3A (5.16– 4.04 p.p.m.), thus establishing the acetyl substituent loca- tion at C3A in the native O-PS. The O-acetyl substitution on residue C (b-D-GlcpNAcA) was indicated to be at position C3C as these 1 Hand 13 C resonances experience similar downfield shifts on de-O-acetylation. A consideration of the experimental evidence thus leads to the full structure of the F. columnare ATCC 43622 LPS native O-chain being an unbranched polymer of a repeating trisaccharide having the structure: ½C½A½B ½! 4Þ-b-d-GlcpNAcA-ð1!4Þ-a-l-FucpNAm-ð1!3Þ-a-d-Sugp-ð1! ½C½A½B ½!4Þ-b-d-GlcpNAcA-ð1!4Þ-a-l-FucpNAm-ð1!3Þ-a-d-Sugp-ð1! 33 "" Ac Ac 3444 L. L. MacLean et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Discussion In this investigation, it was shown by 1D and 2D NMR analysis, MS, and chemical methods that the O-PS of the LPS produced by F. columnare ATCC 43622 is a linear unbranched polymer of trisaccharide units composed of D-GlcNAcA, L-FucNAm and D-Sug having the structure fi4)-b-D-GlcpNAcA-(1fi4)-a-L-FucpNAm-(1fi3)-a-D- Sugp-(1fi, in which the linkage positions and the sequence and pyranoside nature of the glycose residues were estab- lished from NMR analyses. In the native O-PS the D-GlcpNAcA and L-FucpNAm residues were both acetyl- ated at their O3 positions. It is interesting to note that O3-linked D-Sug was found to be a component of the O-PS of the fish pathogen Vibrio ordalii serotype O:2 [15], which is the cause of vibriosis among feral and farmed fish and shellfish. The only other reported bacterial source of this glycose is the specific CPS of Streptococcus pneumoniae type 5 [16]. However, in the latter polysaccharides, the glycose is found in its b- D -configuration in contrast with the a- D -configuration found in the F. columnare O-PS. In agreement with previous studies, we also found that the presence of this 4-ketoglycose in the polymeric structure rendered the O-PS unstable under alkaline conditions and even prolonged storage in aqueous solutions at pH 7. A similar result was found in a study of forbeside C, a saponin of Asterias forbesi [18], which also has a component D-Sug residue. After the precipitation of the LPS from the phenol phase extract of F. columnare cells by ultracentrifugation, a low yield of CPS material was obtained from the ultracentri- fugate by acetone precipitation followed by Sephadex G-50 gel-filtration chromatography, yielding a lipid-free high- molecular-mass void-volume fraction. On analysis, the material proved to have the same structure as the homo- logous LPS O-PS. This material could be considered to be a putative capsule or simply free O-PS. The significance of the O-PS and putative CPS in pathogenesis requires further investigation. In the fish pathogens, Vibrio ordalii O:2 [15] and Vibrio anguillarum O:2 [17], their respective LPS O-PS components and CPSs shared the same respective homo- logous structures, and the same constitution may pertain in F. columnare. Pathogenesis studies have shown a correlation between the capacity of F. columnare to adhere to fish gill epithelium and virulence [6,19,20]. However, the nature of the adhesins involved have not been identified, but possible candidates are LPS, capsule, fimbriae or other appendages of the bacterium, a hypothesis requiring further investigation. It is of note that the structure of the LPS O-antigen of F. columnare differs structurally from the LPS O-antigen of the fish pathogen Flavobacterium psychrophilium [21], which is a linear polymer of a trisaccharide repeating unit composed of L -rhamnose, 2-acetamido-2-deoxy- L -fucose, and 2-N-acetyl-4-N-[(3S,5S)-3,5-dihydroxyhexanoyl]- D - bacillosamine (1 : 1 : 1) [22]. Acknowledgements This work was supported by funding from the Canadian Bacterial Diseases Centres of Excellence Program. We thank Perry Fleming for the large scale production of bacterial cells, and Dr E. Vinogradov for helpful discussions. References 1. Bernardet, J.F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K. & Vandamme, P. (1996) Cutting a Gordian knot: emended classification and description of the genus Flavobacterium,emen- ded description of the Flavobacteriaceae, and proposal of Flavo- bacterium hydatis nom. Nov (basonym, and Cytophaga aquatilis Strohl and Tait 1978). Int. J. Syst. Bacteriol. 46, 128–148. 2.Durborow,R.M.,Thune,R.L.,Hawke,J.P.&Camus,A.C. (1998) Columnaris Disease: a Bacterial Infection Caused by Flavo- bacterium Columnare. Publication no. 479 Southern Regional. Aquaculture Center, Stoneville, MI, USA. 3. Ofek, I. & Doyle, R.J. (1994) In Bacterial adhesion to cells and tissues, pp. 1–16. Chapman & Hall, London. 4. Decostere, A., Haesebrouck, F., Van Driessche, E., Charlier, G. & Ducatelle, R. (1999) Characterization of the adhesion of Flavo- bacterium columnare (Flexibacter columnaris) to gill tissue. J. Fish Dis. 22, 465–474. 5. Decostere, A., Haesebrouch, F. & Derriese, L.A. (1998) Char- acterization of four Flavobacterium columnare (Flexibacter columnaris) strains from tropical fish. Vet Microbiol. 62, 35–45. 6. Decostere, A., Haesebrouck, F., Charlier, G. & Ducatelle, R. (1999) The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphe- nops). Vet. Microbiol. 67, 287–298. 7. Tsai, G.M. & Frasch, C.E. (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Bio- chem. 119, 115–119. 8.Hermansson,K.,Perry,M.B.,Altman,E.,Brisson,J R.& Garcia, M.M. (1993) Structural studies of the O-antigenic poly- saccharide of Fusobacterium necrophorum. Eur. J. Biochem. 212, 801–809. 9. Leontein, K., Lindberg, B. & Lonngren, J. (1978) Assignment of absolute configuration of sugars by g.1.c. of their acetylated gly- cosides formed from chiral alcohols. Carbohydr. Res. 62, 359–362. 10. Johnson, K.G. & Perry, M.B. (1973) Improved techniques for the preparation of bacterial lipopolysaccharides. Can. J. Microbiol. 22, 29–34. 11. Perry, M.B. & Babiuk, L.A. (1983) Structure of the polysaccharide chains of Pasteurella haemolytica (serotype 4) lipopolysaccharide. Biochem. Cell Biol. 62, 108–114. 12. Reinhold, B.B., Hauer, C.R., Plummer, T.H. & Reinhold, V.N. (1995) Detailed structural analysis of a novel, specific O-linked glycan from the prokaryote Flavobacterium meningosepticum. J. Biol. Chem. 270, 13197–13203. 13. Perry, M.B. & Daoust, V. (1973) A synthesis of 2-Amino-2,6-di- deoxy-D-(and L-)galactose (L-Fucosamine) and 2-Amino-2,6-di- deoxy-D-(and L-)talose (Pneumosamine). Can. J. Chem. 51, 974–977. 14. Perry, M.B. & Daoust, V. (1973) A synthesis of 2-Amino- 2,6-dideoxy-L-mannose (L-Rhamnosamine) and 2-Amino- 2,6-dideoxy-L-glucose (L-Quinovosamine). Carbohydr. Res. 27, 460–463. 15. Sadovskaya, I., Brisson, J R., Khieu, N.H., Mutharia, L.M. & Altman, E. (1998) Structural characterization of the lipopoly- saccharide O-antigen and capsular polysaccharide of Vibrio ordalii serotype O: 2. Eur. J. Biochem. 253, 319–327. 16. Jansson, P E., Lindberg, B. & Lindquist, U. (1985) Structural studies of the polysaccharide from Streptococcus pneumoniae type 5. Carbohydr. Res. 140, 101–110. 17. Sadovskaya, I., Brisson, J R., Mutharia, L.M. & Altman, E. (1996) Structural studies of the lipopolysaccharide O-antigen and capsular polysaccharide of Vibrio anguillarum serotype O: 2. Carbohydr. Res. 283, 111–127. Ó FEBS 2003 Flavobacterium columnare polysaccharide (Eur. J. Biochem. 270) 3445 18. Findlay, J.A., Jaseja, M. & Brisson, J R. (1987) Forbeside C, a saponin from Asterias forbesi. Complete structure by nuclear magnetic resonance methods. Can. J. Chem. 65, 2605–2611. 19. Decostere, A. (2002) Flavobacterium columnare infection in fish: the agent and its adhesion to the gill tissue. Verh. K. Acad. Geneeskd. Belg. 64 (6), 421–430. 20. Decostere, A., Ducatelle, R. & Haesebrouck, F. (2002) Flavo- bacterium columnare (Flexibacter columnaris) associated with severe gill necrosis in koi carp (Cyprinus carpio L). Vet. Rec. 150 (22), 694–695. 21. Crump, E.M., Perry, M.B., Clouthier, S.C. & Kay, W.W. (2001) Antigenic characterization of the fish pathogen Flavobacterium psychrophilium. Appl. Environ. Microbiol. 67, 750–759. 22. MacLean, L.L., Vinogradov, E., Crump, E.M., Perry, M.B. & Kay, W.W. (2001) The structure of the lipopolysaccharide O-antigen produced by Flavobacterium psychrophilium (259–93). Eur. J. Biochem. 268, 2710–2716. 3446 L. L. MacLean et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . Structural characterization of the lipopolysaccharide O -polysaccharide antigen produced by Flavobacterium columnare ATCC 43622 Leann L fimbriae or other appendages of the bacterium, a hypothesis requiring further investigation. It is of note that the structure of the LPS O -antigen of F. columnare