Structural studies of the capsular polysaccharide and
lipopolysaccharide O-antigen of
Aeromonas salmonicida
strain
80204-1 produced under
in vitro
and
in vivo
growth conditions
Zhan Wang
1
, Suzon Larocque
1
, Evgeny Vinogradov
1
, Jean-Robert Brisson
1
, Andrew Dacanay
2
,
Marshall Greenwell
2
, Laura L. Brown
2
, Jianjun Li
1
and Eleonora Altman
1
1
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada;
2

-
quinovose, QuiNAc) and having the following structure:
[fi3)-a -
D
-GalpNAcA-(1fi3)-b-
D
-QuipNAc-(1fi4)-b-
D
-
Quip3NAlaNAc-(1-]
n
, where GalNAcA i s partly presented
as an amide and AlaNAc represents N-acetyl-
L
-alanyl
group. CE-ES-MS analysis of CPS and O-chain polysac-
charide confirmed that 40% of GalNAcA was presen t in the
amide form. Direct CE-ES-MS/MS analysis of in vivo cul-
tured cells confirmed the formation of a novel polysaccha-
ride, a structure also formed in vitro, which was previously
undetectable in bacterial cells grown w ithin implants in fish,
and in which GalNAcA was fully amidated.
Keywords: Aeromonas salmonicida; capsular polysaccharide;
lipopolysaccharide; NMR.
Aeromonas salmonicida is the aetiological agent of fu runcu-
losis in s almonid fish, a disease which causes high mort alities
in aquaculture. Considerable effort has been devoted to the
development of e ffective vaccines a gainst furunculosis.
Known extracellular virulence factors of the in vitro-grown
A. salmonicida include surface layer (A-layer) [1], proteases

pathogenesis has not been established.
In the present study we have isolated and c haracterized
the cell-surface carbohydrate antigens of A. salmonicida
Correspondence to E. Altman, Institute for Biological Sciences,
National Research Council of Canada, Ottawa, Ontario,
K1A OR6, Canada. Fax: +1 613 941 1327, Tel.: +1 613 990 0904,
E-mail:
Abbreviations: A-layer, Ae r omonas surfa ce layer; CE- E S-MS, capillary
electrophoresis-electrospray MS; CPS, capsular polysaccharide;
GalNAcA, 2-acetamido-2-deoxy-
D
-galacturonic acid; GalNAcAN,
2-acetamido-2-deoxy-
D
-galacturonamide; LPS, lipopolysaccharide;
Qq-TOF, hybrid quadrupole time-of-flight; TSA, tryptic s oy agar;
TSB, tryptic soy broth.
(Received 18 June 2004, revised 7 September 2004,
accepted 4 October 2004)
Eur. J. Biochem. 271, 4507–4516 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04410.x
strain 80204-1 produced under in vitro growth conditions on
tryptic soy agar (TSA) and in vivo and have demonstrated
that th eir structures are chemically and a ntigenically distinct
from the previously described O-chain polysaccharide [11]
and capsule [13].
Experimental procedures
Bacterial culture conditions
A-layer
–
A. salmonicida avirulent strain 80204-1, a laborat-

(Aldrich, Oakville, O N, Canada). Resultant methyl g ly-
cosides were converted to acetates an d analysed by GLC-
MS using a Hewlett–Packard chromatograph equipped
with a 30 m DB-17 capillary column [180 °C(30min)to
260 °Cat2°CÆmin
)1
] and mass spectra in the electron
impact mode (EI) were recorded using a Varian Saturn
2000 mass spectrometer.
To confirm the ring configuration of constituent sugars,
the LPS sample (20 mg) was dissolved in dry methanol
(5 mL), cooled in dry ice/acetone bath and acetyl chloride
(5 mL) added. The reaction mixture was kept a t 70 °C
for 4 h and dried under a stream of N
2
. M ethanolysis
products were separated by HPLC using a C18 column
(10 · 250 mm, Aqua, Phenomenex Torrance, CA, USA) in
3% MeCN (20 min, isocratic) to 90% MeCN gradient at
3mLÆmin
)1
with the UV detection at 220 nm. Fractions
were dried and analysed by NMR.
Fatty acids were determined by GLC-MS analysis of
their methyl esters derived by sealed-tube hydrolysis of LPS
with 3% (w/v) methanolic hydrogen chloride at 100 °Cfor
16 h and then neutralized with silver carbonate (Aldrich).
For GLC analysis a 30 m DB-5 capillary column [160 °C
(2 min) to 260 °Cat1°CÆmin
)1

polysaccharide was subjected to hydrolysis as described by
Stellner et al. [ 18] a nd methylation analyses were made
according to previously reported conditions for alditol
acetates.
Carboxyl group reduction
Carboxyl group reduction of the CPS and LPS samples
was performed as previously described [19]. Briefly, LPS
(10 mg) was d issolved in distilled water (10 mL) and
following the addition of 1-cyclohexyl-3-(2-morpholino-
ethyl) carbodiimide metho-p-toluenesulfonate (113 mg),
the stirred mixture was maintained at pH 4.7 by titration
with 0.1
M
HCl for 3 h. Following completion of the
reaction a 2
M
solution of sodium borohydride (12.5 mL)
was added slowly a nd the r eaction mixture was main-
tainedatpH7bytitrationwith4
M
HCl. The reaction
wasallowedtoproceedfor2hat22°C, and the solution
was dialysed and lyophilized. The product was purified by
gel permeation chromatography on Sephadex G-100 and
lyophilized (yield 6 mg).
NMR spectroscopy
NMR spectra were performed on Varian INOVA 500 and
600 M Hz spectrometers using standard software. NMR
measurements were made at 25 °Cand60°ConCPSor
LPS samples dissolved in D

3
PO
4
(d
P
0.0 p.p.m.).
Standard homo- and heteronuclear correlated 2D tech-
niques were used for general assignments of the CPS and
LPS O-chain polysaccharide: COSY, TOCSY, NOESY and
HSQC. Due to overlap in proton resonances, selective
TOCSY and TOCSY–TOCSY experiments were used to
complete the assignments [20].
MS
All experiments were performed as described previously in
detail [21]. Briefly, a Crystal Model 310 CE instrument (ATI
Unicam, Cardiff, CA, USA) was coupled to an API 3000 or
Q-Star quadrupole/TOF (Qq-TOF) mass spectrometer
(Applied Biosystems/Sciex, Foster City, CA, USA) via
a microIonspray interface. Sheath solution (isopropanol/
methanol, 2 : 1 ) was delivered at a flow rate of 1 lLÆmin
)1
.
An electrospray stainless steel needle (27 g) w as butted
against the low dead volume tee and enabled the delivery of
the sheath solution t o the end of the capillary column. The
separations were obtained o n  90-cm long bare fused-silica
capillary using 10 m
M
ammonium acetate in deionized
water pH 9.0, containing 5% methanol. A voltage of 2 5 k V

i
pH 7.2, 1 h , 60 °C) and, following the enzyme
deactivation (5 min, 100 °C) and low-speed centrifugation,
lyophilized sample was analysed directly by capillary
electrophoresis-electrospray MS (CE-ES-MS) using
Qq-TOF.
The lyophilized pellet was treated with RNase and
DNasetoreleaseLPS(finalconcentration10lgÆmL
)1
in
0.02
M
ammonium acetate, pH 7.5, 2 h, 37 °C) and lyoph-
ilized following low-speed centrifugation (yield 2 7 m g, dry
weight). It was treated with proteinase K as described above
and bacterial cells were recovered by low-speed centrifuga-
tion. Lyophilized sample was treated with 1% acetic acid
(1 h, 100 °C), desalted using a centrifugal filter device (Pall
Corporation, East Hills, New York, USA) and analysed
directly by CE-ES-MS using a Qq-TOF mass spectrometer.
In addition, lyophilized cells were subjec ted to methanolysis
and methylation analyses as described above for purified
CPSandLPSsamples.
Results
Cells of the A-layer
–
avirulent s train o f A. salmonicida,
80204-1, were grown on TSA, harvested, washed with 2.5%
saline and subjected to the phenol/water extraction [15]
followed by purification of aqueous- and phenol-phase

D
-galactose, 2-amino-2-deoxy-
galactose, 2-amino-2-deoxy-glucose and
L
-glycero-
D
-manno-
heptose [12], were also detected ( 10%). In addition,
phenol-phase soluble LPS was found to contain an a1,6-
linked glucan, as demonstrated by both composition and
methylation analyses. A significant amount of 2-amino-2-
deoxy-
D
-galactose was identified in the hydrolysis products
of both c arboxyl-reduced CPS and LPS [19], confirming the
presence of GalNAcA in the native CPS and LPS. This was
further corroborated by NMR and MS analyses performed
on CPS and carboxyl-reduced LPS. Amino acid analysis
confirmed the presence of
L
-Ala in both polysaccharides.
Fatty acid analysis of both phenol- and aqueous-phase
soluble LPS showed the presence of dodecan oic acid
(C12 : 0), 3-hydroxytetradecanoic acid ( 3-OH C14 : 0),
hexadecanoic acid ( C16 : 0) and 9 -hexadec enoic acid
(C16 : 1n9) as major constituents with 2-hydroxydodeca-
noic acid (2-OH C12 : 0), 3-hyd roxydodecanoic acid (3-OH
C12 : 0) and 9-octadecenoic acid (C18 : 1n9) being minor
components. Fatty acids accounted for 7% (w/w) of the
aqueous-phase LPS and 12% (w/w) of the phenol-phase

3,6-dideoxy-glucose were determined by GLC-MS of the
corresponding (R)-2-bu tyl-glucoside derivatives and found
to be
D
, while the absolute configuration of the 2-acetam-
ido-2-deoxy-galacturonic acid was determined following the
hydrolysis of the carboxyl-reduced CPS and was also found
to be
D
.
The results suggest that A. salmonicida CPS and O-chain
LPS are composed of linear trisaccharide repeating units
containing 3-linked 2-acetamido-2-deoxy-
D
-quinovose,
4-linked 3-[(N-acetyl-
L
-alanyl) amido]-3-deoxy-
D
-quinovose
and 3-linked 2-acetamido-2-deoxy-
D
-galacturonic acid. The
sequence of constituent glycoses and the location of
L
-Ala
were confirmed by 2D NMR analysis performed on both
CPS and aqueous-phase LPS and their methanolysis
products, and CE-ES-MS methods.
In order to sharpen broad resonances due to the extreme

chemical shifts. For residue a only H-1, H -2 (from 2D
COSY) and H-3, H-4 (from 2D TOCSY) (Fig. 2A) could be
identified, due to a small J
4,5
coupling constant which
prevented magnetization transfer past H-4a,suggestinga
galacto configuration. The HSQC
1
H-
13
C experiment
identified C-2 at 49.3 p.p.m. confirming residue a being
2-amino glycose. The H-5a resonance was detected in the
2D NOESY experiment. A high chemical shift of H-4a at
4.43 p.p .m. was typical of a u ronic acid, confirming residue
a to be 2-amino-2-deoxy-a-
D
-galacturonic acid (Table 1)
substituted at position O-3 (C-3a at 77.9 p.p.m. due to a
glycosylation effect [24]). This was further confirmed by
CE-ES-MS and CE-ES-MS/MS analyses performed on
CPS and LPS samples. Several attempts to carry out
HMBC experiments on both CPS and aqueous-phase LPS
samples to confirm the presence of carboxyl group were
unsuccessful, possibly due to a line broadening effect.
For residues b and c it was possible to make partial
assignments of H -1, H -2 ( from 2 D C OSY) and H -3 ( from 2 D
TOCSY). Presence of two methyl groups corresponding to
H-6 of 6-deoxy sugars was observed at 1.27 p.p.m. and
1.29 p.p .m., their H-5 resonances at 3.56 p.p.m. and

TOCSY experiments on samples in 90% H
2
O/10% D
2
O
(Table 1). The NH-2a was assigned on the basis of intra-
residue NOE with H-1a.TheNH-2c was assigned based on
the intraresidue NOEs with H-1c,H-2c and H-3c (Fig. 3B).
The presence of the intraresidue NOEs between the
N-acetyl proton 3b-NH and the C H (2-Ala), CH
3
(3-Ala),
and NH protons of
L
-alanine (NHAc-Ala), confirmed by
NOESY experiment in 90% H
2
O/10% D
2
O, demonstrated
that N-acetyl-
L
-alanyl substituent was located on position 3
of residue b (Fig. 3B).
Proton resonances for the CPS sample did not appear as
resolved multiplets due to heterogeneity resulting from the
presence of both the amide a nd nonamide forms of
GalNAcA and broadening due to high viscosity of the
polysaccharide. Hence, in order to confirm the configur-
ation of the sugars and structure, methanolysis products

fully characterized by 1D NMR a nd 2D COSY and HSQC
experiments. Based on their
1
Hand
13
C chemical shifts
(Table 1), coupling constants and comparison with the
literature values [24], residues b and c were assigned a gluco
configuration.
The sequence of monosaccharides in the repeating unit
of both CPS and O-chain polysaccharide was established
from 2D NOESY spectrum f or anomeric resonances.
Interresidue NOEs were observed between H-1a and H-3c
and between H-1b and H-3a, suggesting a linear struc ture
a-c-b (Fig. 2E,F). In addition, interresidue NOEs between
H-1c and H-4b (F ig. 2G) indicated that residue c was
linked to residue b. This was also supported by results of
Table 1.
1
H- and
13
C-NMR chemical shifts (p.p.m.) for the CPS a nd O-polysaccharide of A. salmonicida strain 80204-1 and its methanolysis products
[1,2]. Spectra were recorded at 6 0 °Cor25°CinD
2
O. For the detection of NH protons spectra were recorded at 25 °C in 90% H
2
O/10% D
2
O
(v/v). The observed

176.0 50.8 19.2 22.7
Product 1
a-GalpNAcA6OMe(1-
a
5.31 4.20 3.93 4.32 4.52 1.97 3.86
100.3 50.9 68.2 70.6 72.6 23.2 54.3
-3)-a-QuipNAc(1-OMe
c
4.66 4.03 3.75 3.42 3.78 1.31 2.05 3.38
99.5 53.8 80.5 77.4 68.8 17.9 23.4 56.4
Product 2
a-Quip3NAlaNAc(1-OMe
b
4.76 3.65 4.00 3.23 3.77 1.28 3.45
AlaNAc 4.34 1.39 2.04
Fig. 2. NMR experiments for assignment and sequence determination of
sugar residues in A. salmonicida CPS and LPS. (A) Proton spectrum at
25 °C. (B) Slice from a 90-ms TOCSY for 1a. (C) 1D TOCSY–TOCSY
(6bc,20ms;5b, 60 ms). (D) 1D TOCSY–TOCSY (6bc,20ms;5c,
90 m s). (E–G) Slice from a 50 ms NOESY for 1 a,1b and 1c reso-
nances. For each se lective experiment the irradiated resonance and the
mixing time are indicated.
Ó FEBS 2004 CPS and LSP O-antigen of A. salmonicida (Eur. J. Biochem. 271) 4511
methylation and CE-ES-MS analyses obtained on both
CPS and LPS.
CPS and the carboxyl-reduced LPS samples of
A. salmonicida were subjected to CE-ES-MS analysis using
an API 3000 as a detector in a positive mode with high
orifice voltage (200 V) that allowed fragmentation of both
polymers (Table 2). Th e CE-ES-MS spectrum of C PS was

anomeric proton resonances of GalNAc and GalNAcAN,
respectively, in the
1
H-NMR spectrum of t he carboxyl-
reduced LPS sample of A. salmonicida (data not shown).
The proposed structure of CPS and O-chain polysaccharide
of A. salmonicida strain 80204-1 is depicted in Fig. 5.
The biological significance of these findings was estab-
lished through the chemical and CE-ES-MS and CE-ES-
MS/MS analyses of t he in vivo cultured A. salmonicida
strain 80204-1 cells [20]. Methanolysis of the bacterial cells
followed by GLC-MS analysis s howed the p resence of
sugars consistent with the composition of CPS and
LPS O-chain polysaccharide, namely 2-amino-2-deoxy-
D
-
quinovose, 3-amino-3-deoxy-
D
-quinovose and 2-amino-
2-deoxy-
D
-galacturonic acid, in the approximate molar
ratio of 1.5 : 1.0 : 0.4. Composition and methylation ana-
lyses o f the inoculum TSB c ulture used to prepare the
growth chambers showed no sugars corresponding to the
proposed novel CPS structure and were c onsistent with
Fig. 3. Partial s tructure of the repeating unit of
A. salmonicida CPS and O-chain polysaccha-
ride with intraresidue NOE connectivities (A)
and 2D NOESY spectrum for A. salmonicida

662.11 661.29 a¢¢-c-b a¢¢-c-b
663.16 662.27 a-c-b –
1297.37 1296.58 – (a¢-c-b)
2
1310.34 1309.58 – a¢¢-c-b-a¢-c-b
1324.23 1323.56 a¢¢-c-b-a-c-b –
1325.18 1324.54 (a-c-b)
2
–
4512 Z. Wang et al.(Eur. J. Biochem. 271) Ó FEBS 2004
the previously identified O-chain containing
L
-rhamnose,
2-amino-2-deoxy-
D
-mannose and
D
-glucose [11], confirming
that the novel CPS and O-chain polysaccharide were
produced during the 72-h in vivo culture p eriod. In addition,
sugars detected in the inoculum TSB culture were also
present. Based on sugar ratios of 3-amino-3-deoxy-
D
-qui-
novose and
L
-rhamnose,  10% of the newly formed CPS
and LPS O-chain polysaccharide was present i n the in vivo
cultured cells. Methylation analysis performed on in vivo
cultured A. salmonicida strain 80204-1 cells confirmed these

CPS. Separation conditions: bare fused-silica
(90 cm · 50 lm i.d., 185 lm o.d), 5% meth-
anol in 15 m
M
ammonium acetate, pH 9.0,
+15 k V. The high orifice voltage of Q-Star
(120 V) pro vided the environm ent to break a
polysaccharide into repeating units, giving
diagnostic fragments arising f rom the cleavage
of glycosidic bonds.
Fig. 5. The pro posed structure of th e C PS and
O-chain polysaccharide of A. salmonicida
strain 80204-1.
Ó FEBS 2004 CPS and LSP O-antigen of A. salmonicida (Eur. J. Biochem. 271) 4513
In order to evaluate the sensitivity of the CE-ES-MS
approach, a series of purified A. salmonicida LPS standards
containing between 100 lgand5lg, were prepared, treated
with 1% acetic acid and subjected to CE-ES-MS analysis
with a high orifice voltage of 120 V (not shown). On the
basis of these experiments it w as established that e ven at t he
lowest dilution corresponding to 5 lg of purified LPS,
the observed CE-ES-MS spectrum was consistent with the
previously established fragmentation pattern, where
the fragment ion at m/z 663.3 corresponded to the mass
of one trisaccharide repeating unit-containing species with
GalNAcA, fragment ion at m/ z 1325.5 corresponded to the
mass of two t risaccharide repeating units, and fragmen t ion
at m/z 1067.4 was consistent with the loss of Qui3NAlaNAc.
Due to a relatively high background at lower dilutions of the
LPS standard and i n o rder to con firm the CE-ES-MS

80204-1 produced under in vitro growth conditions on TSA.
Both polysaccharides were shown by c omposition, methy-
lation analysis, NMR and MS methods to be composed of
linear t risaccharide repeating units containing 3-linked
2-acetamido-2-deoxy-
D
-quinovose, 4-linked 3-[(N-acetyl-
L
-alanyl)amido]-3-deoxy-
D
-quinovose and 2-acetamido-
2-deoxy-
D
-galacturonic acid. To our knowledge, this is
the first report describing the presence of the CPS in
A. salmonicida.
We have confirmed by direct CE-ES-MS analysis that
both CPS and O-chain polysaccharide were also present in
the in vivo -grown cells of A. salmonicida strain 80204-1
harvested at 72 h postimplant surgery. These polysaccha-
rides were not detected in the in vitro-grown bacterial
inoculum TSB culture used for the implants.
To date a limited number of bacteria have been reported
to produce capsular and O-chain polysaccharides with
identical structures. It appears that this property is not
uncommon for fish pathogens and we have previously
reported similar findings for Listonella (formerly Vibrio)
anguillarum and V. ordalii [26,27]. It should be noted that
the structures of the CPS and O-chain polysaccharide
of L. anguillarum and V. ordalii have recen tly been

may b e critical for formation of the polysaccharide [22]. The
devised microanalytical procedure is suitable for direct
analysis of both CPS and LPS and could detect as little as
2 lgofLPSinthesample.
The present studies suggest that caution s hould be
exercised when in vitro -cultured cells are used for isolation
and structural analysis of bacterial polysaccharides as the
resultant structural i nformation may not be biologically
relevant to in vivo conditions. Application of CE-ES-MS
methods for direct analysis of cells from experimental
models of infection can overcome these limitations. CE-ES-
MS has been proven to b e a power ful technique to
distinguish different glycoforms when applied to lipopoly-
saccharides from Neisseria meningitidis [28]. However, in
this particular application we a ttempted to improve the
detection sensitivity by separating the C PS and LPS
from salts and other matrices associated with the sample
preparation.
These findings suggest that additional viru lence factors
such as CPS and novel LPS O-chain polysaccharide
contribute to the pathogenesis of A. salmonicida in vivo
and emphasize a critical role of a host in host–pathogen
interactions.
Acknowledgements
We thank Dr Giles Olivier from the Gulf Sciences Centre, Department
of Fishe ries and Oceans Canada, Moncton, NB for the kind gift of
A. salmonicida strains used in this study, Dr Jessica Boyd from the
Institute o f Marine Biosciences, National Research Council of Canada
for providing A. salm onicida stock cultures and the Dalhousie
University Aquatron for wetlab s pace. We also thank Mrs Vandana

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