O-PS LPS 1.40 g was delipidated by treatment with 1% v/v acetic acid 100 mL at 100C for 2 h and, after removal of precipitated lipid A 170 mg, the lyophilized water-soluble products were
Trang 1Structural characterization of the lipopolysaccharide O -polysaccharide
Leann L MacLean1, Malcolm B Perry1, Elizabeth M Crump2and William W Kay2
1
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada;2Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada
The structure of the antigenic O-chain polysaccharide of
Flavobacterium columnareATCC 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 columnarisor 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
16C in a 52-L fermentor in medium of composition: tryptone, 4 g; yeast extract, 0.4 g; MgSO4, 0.5 g; CaCl2, 0.5 g; sodium acetate, 0.2 g; maltose, 10 gÆL)1; pH was adjusted to 7.00 with 0.1M NaOH A 2.5-L inoculum grown at 22C 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 4C) in late exponential phase at 25.5 h growth (A600¼ 3.34) After acidification with acetic acid to pH 4 at 0C to break the gel-like constitution, the suspended cells were harvested by centrifugation (yield
300 g wet paste)
Preparation of LPS andO -PS
F columnare cells (300 g wet paste) were extracted for
15 min at 65C with vigorously stirred 50% (w/v) aqueous phenol (1.2 L), and, after cooling (4C) 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 (0.02M, pH 7.0, 80 mL) and then treated sequentially with RNase, DNase and proteinase K (37C, 2 h each) The digests were cleared by low-speed centrifugation (4000 g) and then subjected to ultracentrifugation (105 000 g, 12 h, 4C) 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!
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)
Trang 2dissolved 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 100C 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 (Kav0.03–0.12,
390 mg) and a low-molecular-mass putative core
oligosac-charide fraction (Kav0.75, 110 mg)
Chromatography and electrophoresis
Descending preparative paper chromatography was
per-formed on water-washed Whatman 3MM 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 (RGN) 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 170C (delay 4 min) to
240C at 4 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
(TG¼ 1.00)
Polysaccharide was separated by Sephadex G-50 column
(2· 85 cm) chromatography using 0.05M 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
1H and 13C NMR spectra were recorded on a Varian
Inova 400 spectrometer with samples in 99% D2O at
55C, and internal acetone standard (2.225 p.p.m for
1H and 31.07 p.p.m for 13C) 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, 70C) 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 (47 mg) in water (10 mL) with 1-(3-dimethylaminopropyl)-3-ethyl carbodi-imide (150 mg) maintained at pH 4.7 over 4 h followed by reduction at 0C 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 NaBH4 under the same experimental conditions
General methods Hydrolyses were carried out in sealed tubes with either 6M
HCl (100C, 3 h) or 2Mtrifluoroacetic acid (105C, 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 (NaBD4or NaBH4) and acetylation (Ac2O) 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 20C 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 (Kav0.7,
180 mg) which gave a strong colorimetric (phenol/H2SO4) 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 2M trifluoroacetic acid (105C, 4 h) O-PS hydrolysis products gave a low yield ( 2%) mixture of mannose, galactose andL-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
Trang 3The 1D 1H-NMR spectrum of the O-PS showed inter
alia: three anomeric glycose H1 proton signals at 5.14 (J1,2
2.2 Hz), 4.97 (J1,2 3 Hz) and 4.70 (J1,28.8 Hz) p.p.m with
J1,2couplings 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 13C-NMR spectrum of the O-PS (Fig 1) showed
inter aliathree anomeric signals at 102.6 (JC-1,H-1164 Hz),
97.1 (JC-1,H-1 172 Hz) and 97.0 (JC-1,H-1 180 Hz) p.p.m
having JC-1,H-1coupling constants indicative of one
b-link-age and two a-linkb-link-ages, 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 (CH3-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 1H-NMR and
13C-NMR spectra and the characterization of the glycose components in the O-PS were determined from the appli-cation of COSY, TOCSY, NOESY and1H,13C-HSQC and HMBC experiments (Table 1, Fig 2) For the analysis,
Fig 1.13C-NMR spectrum of F columnare O-PS recorded at 55 °C (125 MHz).
Table 1. 1Hand13C NMR chemical shifts of the native LPS O-PS from F columnare ATCC 43622 Spectra run in D 2 O at 55 C with internal acetone reference (2.225 p.p.m for1H and 31.07 p.p.m for13C) Coupling constants (Hz) are given in parentheses Tentative assignments for residue A: N-H7 (8.83 p.p.m.) and N-H71(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
Trang 4glycose 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 (H4,5 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 (JHl,2 2 Hz, JC-1,H-1172 Hz)
From NMR data, residue C was assigned the
b-gluco-pyranose configuration from its observed large ring region
JH,Hcoupling constants for J2,3, J3,4and J4,5( 10 Hz), and
from its anomeric coupling constants, JC-1,H-1164 Hz, and
J1,28.8 Hz The correlation of H2C to the C2C at 55.4 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
of 3 Hz and JC-1,H-1of 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
1H-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 (RGN1.47) from its specific optical rotation {[a]D)81 (c 0.2, water) Lit [a]D– 95 [13]}, the identity of its1 H-NMR spectrum with that of an authentic sample, and the fact that its reduced (NaBD4) and acetylated product on GLC/MS gave a single peak corresponding in retention time (TG0.93) and mass spectrum to an authentic sample
of 1,3,4,5,-tetra-O-acetyl-2-acetamido-2,6-dideoxy-D -galact-itol-[1-2H]
The hexuronic acid component C was identified as 2-acetamido-2-deoxy-D-glucuronic acid from the isolation
Fig 2.1H-13C H SQC shift correlation map
of the spectral regions1H(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.
Trang 5of 2-amino-2-deoxy-D-glucose-[6-2H2], from the hydrolysis
product of the reduced (NaBD4) carbodiimide-activated
O-PS The latter glycose isolated by preparative paper
chromatography (RGN 1.00) was identified by GLC/MS
of its reduced (NaBD4) acetylated derivative
1,3,4,5,6-penta-O-acetyl-2-acetamido-2-deoxyglucitol-[1-2H, 6-2H2]
(TG 1.22), and its D-configuration was confirmed from
the specific optical rotation of its hydrochloride derivative
{[a]D+ 67 (c 0.3, 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 (NaBD4)
carbodiimide-activated O-PS also yielded two separated
aminoglycose fractions identified as a mixture of
2-amino-2,6-dideoxy-D-(andL)galactose {RGN1.48; [a]D)4 (c 0.2,
water) [13]} and 2-amino-2,6-dideoxy-D-glucose {RGN1.83;
[a]D+ 52 (c 0.4, water); Lit [a]D+ 50 [14]}, in
approximately equal yield GLC/MS of their individual
reduced (NaBH4) and acetylated alditiol derivatives gave
single peaks corresponding in retention times to
1,3,4,5-tetra-O-acetyl-2-acetamido-2,6-dideoxygalactitol (TG 0.93)
and 1,3,4,5-tetra-O-acetyl-2-acteamido-2,6-dideoxyglucitol
(TG0.90) 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 theD-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 theD-configuration assigned to residue
B Preparative paper chromatographic separation of the
hydrolysis products of NaBH4-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 1H-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 respectiveD-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-PS were estab lished from 1D and 2D NOE and long-range multiple-bond 1H-13C (HMBC) correlations experiments Interresidue NOEs were seen from H1B to H4C and to its own H2B, from H1A to H2A and across the glycosidic bond to H3B, and also from 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, from C1C (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
asfi4)-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, and C4C 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 (50C, 1 h) resulted in the 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 substituloca-tion on residue C (b-D-GlcpNAcA) was indicated to be at position C3C as these 1H and 13C resonances experience similar downfield shifts on de-O-acetylation A consideration of the experimental evidence thus leads to the full structure of the
F columnareATCC 43622 LPS native O-chain being an unbranched polymer of a repeating trisaccharide having the structure:
½! 4Þ-b-d-GlcpNAcA-ð1!4Þ-a-l-FucpNAm-ð1!3Þ-a-d-Sugp-ð1!
½!4Þ-b-d-GlcpNAcA-ð1!4Þ-a-l-FucpNAm-ð1!3Þ-a-d-Sugp-ð1!
Trang 6In 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 columnarediffers 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 Durb orow, 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 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 polylipopoly-saccharide 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.
Trang 718 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.