salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide.. Direct CE-ES-MS/MS analysis of in vi
Trang 1Structural studies of the capsular polysaccharide and
Zhan Wang1, Suzon Larocque1, Evgeny Vinogradov1, Jean-Robert Brisson1, Andrew Dacanay2,
Marshall Greenwell2, Laura L Brown2, Jianjun Li1and Eleonora Altman1
1
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada;2Institute for Marine Biosciences, National Research Council of Canada, Halifax, Canada
Aeromonas salmonicidais a pathogenic aquatic bacterium
and the causal agent of furunculosis in salmon In the course
of this study, it was found that when grown in vitro on
tryptic soy agar, A salmonicida strain 80204-1 produced a
capsular polysaccharide with the identical structure to that
of the lipopolysaccharide O-chain polysaccharide A
com-bination of 1D and 2D NMR methods, including a series of
1D analogues of 3D experiments, together with capillary
electrophoresis-electrospray MS (CE-ES-MS),
composi-tional and methylation analyses and specific modifications
was used to determine the structure of these polysaccharides
Both polymers were shown to be composed of linear
trisaccharide repeating units consisting of
2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA),
3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose{3-[(N-acetyl-L-alanyl)
amido]-3-deoxy-D-quinovose, Qui3NAlaNAc} and
2-ace-tamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D -quinovose, QuiNAc) and having the following structure: [fi3)-a-D-GalpNAcA-(1fi3)-b-D-QuipNAc-(1fi4)-b-D -Quip3NAlaNAc-(1-]n, where GalNAcA is 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 present 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 within implants in fish, and in which GalNAcA was fully amidated
Keywords: Aeromonas salmonicida; capsular polysaccharide; lipopolysaccharide; NMR
Aeromonas salmonicidais the aetiological agent of
furuncu-losis in salmonid fish, a disease which causes high mortalities
in aquaculture Considerable effort has been devoted to the
development of effective vaccines against furunculosis
Known extracellular virulence factors of the in vitro-grown
A salmonicidainclude surface layer (A-layer) [1], proteases
[2], haemolysins [3], type IV pili [4] and LPS [5] Very little is
known about the role of these factors in vivo and their role in
furunculosis The A-layer is believed to play a crucial role
in the bacterial protection against complement-mediated
killing in vitro and contributes to bacterial survival in
phagocytes or host tissues [6] In addition to the A-layer,
lipopolysaccharide (LPS) is another at least partially exposed cell surface antigen that appears to mediate invasion [7] Monoclonal and polyclonal antibody analysis
of a variety of A salmonicida strains has shown that like the A-layer, the surface-exposed regions of LPS are antigeni-cally cross-reactive [8] Formation of capsular polysaccha-ride (CPS) covering the A-layer has been reported to be produced during the in vivo culture of A salmonicida in surgically implanted intraperitoneal culture chambers [9] Moreover, Merino et al [10] have reported that when grown under conditions promoting capsule formation, strains of A salmonicida exhibited significantly higher ability to invade fish cell lines It suggests that, as with the A-layer and LPS, CPS is an important virulence factor, essential for host cell invasion and bacterial survival Previous studies have determined the structure of the O-chain polysaccharide of A salmonicida strain SJ-15 [11] Partial structure of the core oligosaccharide from the same strain of A salmonicida was also determined [12] In both instances A salmonicida strain SJ-15 was cultured in tryptic soy broth (TSB) at 25C In addition, other reports des-cribe capsular material isolated from cells grown on yeast extract-peptone-glucose-mineral salts [13] The relevance of these structures to in vivo-cultured bacteria and their role in pathogenesis has not been established
In the present study we have isolated and characterized 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: eleonora.altman@nrc-cnrc.gc.ca
Abbreviations: A-layer, Aeromonas surface layer; CE-ES-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 soy agar;
TSB, tryptic soy broth.
(Received 18 June 2004, revised 7 September 2004,
accepted 4 October 2004)
Trang 2strain 80204-1 produced under in vitro growth conditions on
tryptic soy agar (TSA) and in vivo and have demonstrated
that their structures are chemically and antigenically distinct
from the previously described O-chain polysaccharide [11]
and capsule [13]
Experimental procedures
Bacterial culture conditions
A-layer–A salmonicidaavirulent strain 80204-1, a
laborat-ory-derived mutant of strain 80204 obtained by subculture
[14], was grown on TSA plates The inoculum was cultured
in TSB (Difco Laboratories, Detroit, MI, USA) at 15C
until it reached D600of 0.17 and spread across the surface of
TSA plates The plates were incubated at 15C for 5 days,
washed with 0.01MNaCl/PipH 7.4 and subjected to a low
speed centrifugation (3000 g, 4C, 10 min) The cells were
killed with in 1% (w/v) phenol solution (4 h, 22C) and
harvested by centrifugation (yield 25 g, wet weight)
Isolation and purification of CPS and LPS
The cells were washed with 2.5% saline (w/v) and extracted
by the method of Westphal et al [15] Phenol and water
layers were collected separately, dialysed against tap water
and lyophilized The lyophilizates were redissolved in 1%
saline (w/v) and the clear solution was subjected to
ultracentrifugation (105 000 g, 4C, 10 h); the LPS pellets
were redissolved in water and lyophilized Clear supernatant
was dialysed until salt-free, lyophilized and used for
isolation of CPS Pure CPS was obtained by gel filtration
on Sephadex G-100 column (yield 12 mg)
Analytical methods
CPS and LPS samples (0.5 mg) were hydrolysed with 3%
(w/v) methanolic hydrogen chloride at 85C for 5 h and
the reaction mixture was neutralized with silver carbonate
(Aldrich, Oakville, ON, Canada) Resultant methyl
gly-cosides were converted to acetates and analysed by
GLC-MS using a Hewlett–Packard chromatograph equipped
with a 30 m DB-17 capillary column [180C (30 min) to
260C at 2 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 at 70C
for 4 h and dried under a stream of N2 Methanolysis
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
3 mLÆmin)1with 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 100C for
16 h and then neutralized with silver carbonate (Aldrich)
For GLC analysis a 30 m DB-5 capillary column [160C
(2 min) to 260C at 1 CÆmin)1] was used and the identity
of each fatty acid was established by comparison of its MS with that of the reference compound
The absolute configuration of glycoses was established by capillary GLC of their acetylated (-)-2-butyl glycosides, according to the method of Leontein et al [16] The identity
of each glycose derivative was established by comparison of its GLC retention time and MS with that of an authentic reference sample The synthetic
2-acetamido-2-deoxy-L-quinovose was a gift from M B Perry (National Research Council, Ottawa, ON, Canada) The absolute configuration
of the 2-acetamido-2-deoxy-galacturonic acid was deter-mined following the hydrolysis of the carboxyl-reduced CPS
Presence of L-alanine was confirmed by amino acid analysis For this, 0.6 mg of CPS was hydrolysed in 5.7M hydrochloric acid containing 0.1% phenol (w/v) for 1 h at
160C under vacuum The acid was removed by vacuum centrifugation (speed-vac) with NaOH trap and the con-centrated sample was injected into the amino acid analyser based on the cation exchange chromatography with amino acid detection by ninhydrin colour at 570 nm except for proline at 440 nm
Methylation analysis The CPS and LPS samples were methylated according
to the method of Ciucanu & Kerek [17] Methylated polysaccharide was subjected to hydrolysis as described by Stellner et al [18] and 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 dissolved 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 0.1M HCl for 3 h Following completion of the reaction a 2Msolution of sodium borohydride (12.5 mL) was added slowly and the reaction mixture was main-tained at pH 7 by titration with 4M HCl The reaction was allowed to proceed for 2 h at 22C, 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 MHz spectrometers using standard software NMR measurements were made at 25C and 60 C on CPS or LPS samples dissolved in D2O at pD 6.5 at concentrations
of 6 mgÆmL)1 for CPS or 20 mgÆmL)1for LPS For the detection of NH protons spectra were recorded at 25C in 90% H2O/10% D2O (v/v)
All NMR experiments were performed using a 5-mm or 3-mm indirect detection probe with the1H coil nearest to the sample The observed1H chemical shifts are reported relative to external acetone (2.225 p.p.m.), and the 13C
Trang 3chemical shifts are quoted relative to the methyl group of
external acetone (31.07 p.p.m.)
31P-NMR experiments were performed on a Varian
INOVA 200 MHz spectrometer, chemical shifts are
given relative to the external 85% H3PO4(dP 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) was butted
against the low dead volume tee and enabled the delivery of
the sheath solution to the end of the capillary column The
separations were obtained on 90-cm long bare fused-silica
capillary using 10 mM ammonium acetate in deionized
water pH 9.0, containing 5% methanol A voltage of 25 kV
was typically applied at the injection The outlet of the
capillary was tapered to 15 lm internal diameter using a
laser puller (Sutter Instruments, Novato, CA, USA) Mass
spectra were acquired with an orifice voltage of +120 V
Fragment ions formed by collision activation of selected
precursor ions with nitrogen in the reference frame-only
quadrupole collision cell, were registered by mass and
analysed by TOF
Analysis of thein vivo cultured A salmonicida strain
80204-1 cells
In vivoculture was performed using ligated dialysis tubing
bags filled with bacterial suspension of A salmonicida strain
80204-1, surgically implanted in the peritoneal cavity of
juvenile Atlantic salmon, and harvested at 72 h postsurgery
as in Dacanay et al [22] Bacterial cells, 2.5· 1011colony
forming units, were washed with 2.5% (w/v) saline, and the
pellet recovered by low-speed centrifugation (3000 g, 4C,
10 min) and lyophilized The supernatant was dialysed
against distilled water and lyophilized It was treated with
proteinase K (final concentration 250 lgÆmL)1 in 0.01M
NaCl/Pi pH 7.2, 1 h, 60C) and, following the enzyme
deactivation (5 min, 100C) 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
DNase to release LPS (final concentration 10 lgÆmL)1in
0.02Mammonium acetate, pH 7.5, 2 h, 37C) and
lyoph-ilized following low-speed centrifugation (yield 27 mg, 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, 100C), 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 subjected to methanolysis and methylation analyses as described above for purified CPS and LPS samples
Results
Cells of the A-layer– avirulent strain of 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 soluble LPS by ultracentrifugation Crude CPS was recov-ered from the initial 2.5% saline wash of bacterial cells and from 1% saline wash of the aqueous- and phenol-phase soluble LPS and purified by gel permeation chromatogra-phy on Sephadex G-100 column CPS eluted as a broad peak in a void volume of a Sephadex G-100 column Fractions were collected and analysed colorimetrically for aldose [23] Three fractions, designated FI–FIII (not shown), were pooled and analysed by NMR Fraction FI was contaminated by an a1,6-glucan while fractions FII and FIII were homogeneous yielding a glucan-free CPS that was used for further analysis
Methanolysis of CPS with 3% methanolic hydrogen chloride, followed by acetylation and GLC analysis of the resultant methyl glycosides afforded 2-amino-2,6-dideoxy-glucose, 3-amino-3,6-dideoxy-glucose and 2-amino-2-deoxy-galacturonic acid in the approximate molar ratio of 1.0 : 1.0 : 0.9 LPS O-chain components previously identi-fied by Shaw et al [11] and produced when A salmonicida was cultured in TSB, namely L-rhamnose, 2-amino-2-deoxy-D-mannose andD-glucose, and core oligosaccharide components, D-glucose, D-galactose, 2-amino-2-deoxy-galactose, 2-amino-2-deoxy-glucose andL-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 carboxyl-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 ofL-Ala in both polysaccharides Fatty acid analysis of both phenol- and aqueous-phase soluble LPS showed the presence of dodecanoic acid (C12 : 0), 3-hydroxytetradecanoic acid (3-OH C14 : 0), hexadecanoic acid (C16 : 0) and 9-hexadecenoic acid (C16 : 1n9) as major constituents with 2-hydroxydodeca-noic acid (2-OH C12 : 0), 3-hydroxydodeca2-hydroxydodeca-noic 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 LPS suggesting an under-acylation pattern The31P-NMR spectrum of aqueous-phase LPS in D2O, pH 6.5 showed two distinct groups of resonances, indicating the presence of phosphate monoester at 0.49 p.p.m and phosphate diester centred at)1.79 p.p.m
Methylation analysis of the carboxyl-reduced CPS and subsequent GLC-MS analysis revealed the presence
of 2,6-dideoxy-4-O-methyl-2-(N-methylacetamido)-glucose,
Trang 43,6-dideoxy-2-O-methyl-3-(N-methylacetamido)-glucose and
2-deoxy-2-(N-methylacetamido)-4,6-di-O-methyl-galactose
in the approximate molar ratio of 0.8:1.0: 0.8, while
GLC-MS analysis of the native CPS showed the presence of
2,6-dideoxy-4-O-methyl-2- (N-methylacetamido)-glucose
and
3,6-dideoxy-2-O-methyl-3-(N-methylacetamido)-glu-cose only, in approximate molar ratio of 0.5 : 1.0, and
was consistent with the presence of GalNAcA in the native
CPS An additional minor component was also detected in
GLC-MS analysis of both the native and the
carboxyl-reduced CPS, its mass spectrum consistent with that of
3,6-dideoxy-2-O-methyl-3-[(N-acetyl-L-alanyl)
methylami-do]-glucose, suggesting that L-Ala was located on
3-amino-3,6-dideoxy-glucose The absolute configurations
of 2-acetamido-2,6-dideoxy-glucose and
3-acetamido-3,6-dideoxy-glucose were determined by GLC-MS of the
corresponding (R)-2-butyl-glucoside derivatives and found
to beD, 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 beD
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 ofL-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
viscosity of the CPS sample in water, some 2D NMR
experiments were carried out on a CPS sample dissolved in
D2O, pD 6.5 at 60C Due to the structural identity of CPS
and O-chain polysaccharide based on their compositional
and methylation analyses, 1D1H-NMR spectra (Fig 1,B)
and a good solubility of LPS probably attributable to a
relatively low fatty acid content, aqueous-phase LPS was
used in most of 1D analogues of 2D NMR experiments
The 1D 1H-NMR spectrum of CPS showed three
reso-nances in the low-field region (5.3–4.4 p.p.m.) in the relative
ratio of 1 : 1 : 3, of which three were later attributed to resonances from anomeric protons by direct correlation with corresponding13C resonances in a 2D heteronuclear
1H)13C correlation HSQC spectrum
Initial assignments were made from 2D COSY and TOCSY experiments carried out on the CPS sample (Table 1) From the HSQC spectrum in Fig 1C, the anomeric protons of three sugar residues were designated a–c, according to the decreasing order of their proton 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 J4,5 coupling constant which prevented magnetization transfer past H-4a, suggesting a galacto configuration The HSQC 1H-13C 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 uronic 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 2D COSY) and H-3 (from 2D 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 3.34 p.p.m., respectively, could be traced through 2D COSY connectivities In order to assign unambiguously the reso-nances for residues b and c selective 1D TOCSY–TOCSY experiments were carried out with selective excitation of both H-6b and H-6c in the first step followed by selective excitation
of H-5c and H-5b, respectively, permitting complete assign-ment of resonances for residues b and c (Table 1, Fig 2C,D) Assignments of13C resonances were carried out by direct correlation of 1H resonances with 13C resonances in a heteronuclear1H-13C HSQC experiment (Table 1, Fig 1C)
Fig 1 1 H-NMR spectra for CPS (A), aque-ous-phase LPS (B) and HSQC spectrum for
A salmonicida CPS (C), recorded at
600 MHz, 60 °C, pD 6.5.
Trang 5Most NH resonances of CPS and O-chain polysaccharide
were assigned on the basis of their large coupling constants
with the ring protons H-C-N-H (9–10 Hz) via COSY and
TOCSY experiments on samples in 90% H2O/10% D2O
(Table 1) The NH-2a was assigned on the basis of
intra-residue NOE with H-1a The NH-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 CH (2-Ala), CH3(3-Ala),
and NH protons of -alanine (NHAc-Ala), confirmed by
NOESY experiment in 90% H2O/10% D2O, 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 and 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 were purified and analysed by NMR to extract coupling constants LPS sample was subjected to methanolysis and resultant methyl glycosides separated on a C18 column Fractions were analysed by 1H-NMR and methyl 3-[(N-acetyl-L-alanyl) amido]-3,6-dideoxy-a-D -glucopyrano-side, methyl 2-acetamido-2,6-dideoxy-a-D-glucopyranoside, and two disaccharides, in both methyl a- and b-glycoside form, could be readily identified A number of other products were separated as a mixture and these were not analysed further Two products, namely methyl 3-O-(methyl-2-acetamido-2-deoxy-a-D -galactopyra-nosyluronate)-2-acetamido-2,6-dideoxy-a-D -glucopyr-anoside (Product 1) and methyl 3-[(N-acetyl-L-alanyl) amido]-3,6-dideoxy-a-D-glucopyranoside (Product 2), were fully characterized by 1D NMR and 2D COSY and HSQC experiments Based on their 1H and 13C 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 for anomeric resonances Interresidue NOEs were observed between H-1a and H-3c and between H-1b and H-3a, suggesting a linear structure a-c-b (Fig 2E,F) In addition, interresidue NOEs between H-1c and H-4b (Fig 2G) indicated that residue c was linked to residue b This was also supported by results of
Table 1.1H- and13C-NMR chemical shifts (p.p.m.) for the CPS and O-polysaccharide of A salmonicida strain 80204-1 and its methanolysis products [1,2] Spectra were recorded at 60 C or 25 C in D 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 1 H and 13 C chemical shifts are reported relative to external acetone (d H 2.225 p.p.m., d C 31.07 p.p.m.) Error for d H ¼ 0.02 p.p.m and error for d C ¼ 0.2 p.p.m.
CPS and O-polysaccharide
-3)-a-GalpNAcA(1-a
5.22 4.32 3.95 4.43 4.12 1.99 8.30 99.8 49.3 77.9 71.0 72.9 23.2
-3)-b-QuipNAc(1-c
4.43 3.65 3.59 3.29 3.34 1.29 1.99 8.69 102.0 55.7 81.4 77.4 72.6 17.9 23.2
-4)-b-Quip3NAlaNAc(1-b
4.55 3.33 3.82 3.35 3.56 1.27 8.04 105.2 72.5 56.4 80.5 73.3 17.9
Product 1
a-GalpNAcA6OMe(1-a
-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
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, 20 ms; 5b, 60 ms) (D) 1D TOCSY–TOCSY (6bc, 20 ms; 5c,
90 ms) (E–G) Slice from a 50 ms NOESY for 1a, 1b and 1c
reso-nances For each selective experiment the irradiated resonance and the
mixing time are indicated.
Trang 6methylation and CE-ES-MS analyses obtained on both
CPS and LPS
CPS and the carboxyl-reduced LPS samples of
A salmonicidawere 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) The CE-ES-MS spectrum of CPS was
consistent with the presence of two major species at m/z 663
and m/z 1326, corresponding to one and two trisaccharide
repeating unit-containing species, respectively, with the
molecular masses for constituent sugar residues being m/z
258 for Qui3NAlaNAc, m/z 217 for GalNAcA and m/z 187
for QuiNAc In addition, fragments at m/z 405 and m/z
1067 consistent with the loss of Qui3NAlaNAc were also
observed The CE-ES-MS spectrum of the carboxyl-reduced LPS showed the presence of an additional fragment
at m/z 649, a product expected following the carboxyl group reduction of GalNAcA and corresponding to the CPS trisaccharide repeating unit containing 2-acetamido-2-deoxy-galactose residue Presence of a minor component
at m/z 662 was unexpected and suggested that some of carboxyl groups in GalNAcA were substituted by a primary amine In order to confirm the extent of amidation of GalNAcA residue, the CPS sample was analysed by
CE-ES-MS using a Qq-TOF mass spectrometer The extent of amidation could be determined by the intensity ratio of fragments at m/z 662.2 Da (GalNAcAN-containing trisac-charide repeating unit) and m/z 663.2 Da (GalNAcA-containing trisaccharide repeating unit) (Fig 4A, inset), based on this ratio about 40% of GalNAcA in both CPS and O-chain polysaccharides was present in the form of a primary amine When the carboxyl-reduced A salmonicida LPS was subjected to the same analysis, as expected, the ion
at m/z 663.2 disappeared giving rise to a new abundant ion
at m/z 649.2, corresponding to the product following the carboxyl-reduction of GalNAcA (Fig 4B, inset) which was also consistent with the signal intensities obtained for anomeric proton resonances of GalNAc and GalNAcAN, respectively, in the 1H-NMR spectrum of the 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 MS and CE-ES-MS/MS analyses of the in vivo cultured A salmonicida strain 80204-1 cells [20] Methanolysis of the bacterial cells followed by GLC-MS analysis showed the presence 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 of the inoculum TSB culture used to prepare the growth chambers showed no sugars corresponding to the proposed novel CPS structure and were consistent with
Fig 3 Partial structure 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 LPS (B) acquired with a mixing time of 100 ms
in 90% H 2 O/10% D 2 O The proton spectra are also shown along both axes with assign-ments.
Table 2 Positive ion CE-ES-MS data and proposed composition of the
native CPS and the carboxyl-reduced O-chain polysaccharide of
A salmonicida strain 80204-1 Monoisotope mass units were used for
calculation of molecular mass values based on proposed compositions
as follows: GalNAcA, 217.06; GalNAc, 203.08; GalNAcAN, 216.08;
QuiNAc, 187.09; Qui3NAc, 187.09; Ala, 71.04 For a, b and c
desig-nation see Table 1 a¢ ¼ GalNAc; a¢¢ ¼ GalNAcAN.
Observed
ion (m/z)
Caculated
mass (Da)
Proposed composition
CPS
Carboxyl-reduced O-chain polysaccharide 204.04 203.08 – a¢
217.03 216.08 a¢¢ a¢¢
391.11 390.17 – a¢-c
404.11 403.17 a¢¢-c a¢¢-c
405.08 404.15 a-c –
649.17 648.29 – a¢-c-b
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 –
Trang 7the previously identified O-chain containing L-rhamnose,
2-amino-2-deoxy-D-mannose andD-glucose [11], confirming
that the novel CPS and O-chain polysaccharide were
produced during the 72-h in vivo culture period In addition,
sugars detected in the inoculum TSB culture were also
present Based on sugar ratios of 3-amino-3-deoxy-D
-qui-novose andL-rhamnose, 10% of the newly formed CPS
and LPS O-chain polysaccharide was present in the in vivo
cultured cells Methylation analysis performed on in vivo
cultured A salmonicida strain 80204-1 cells confirmed these
findings and showed the presence of
2,6-dideoxy-4-O-methyl-2-(N-methylacetamido)-glucose and
3,6-dideoxy-2-O-methyl-3-(N-methylacetamido)-glucose, in accordance
with the proposed structure of CPS The linkage of
GalNAcA could not be verified as the methylation analysis
was carried out without the carboxyl group reduction step
To confirm the sequence of this newly formed
polysac-charide detected in the in vivo cultured cells, CE-ES-MS
analysis was carried out on both the concentrated bacterial cell saline wash containing CPS and on the bacterial pellet following mild acid hydrolysis to release delipidated LPS Initially, the CPS and LPS samples were subjected to CE-ES-MS analysis with the typically used orifice of 45 V The mass spectra indicated no individually separated ion peaks and showed only a broad peak (data not shown) corresponding to a range of molecular masses which could
be attributed to CPS or LPS O-chain polysaccharide repeating unit fragments of different length Previously,
we have successfully applied this approach to the partial degradation of the polysaccharide into shorter oligosaccha-ride units due to front-end collision induced dissociation [25] It is noteworthy to mention that this technique might not be suitable for determination of molecular masses of intact CPS and LPS O-chain polysaccharide but the information provided by this pseudo pyrolysis MS could
be is very useful for characterization of their repeating units
Fig 4 CE-ES-MS analysis (+ ion mode) of
LPS (A) and carboxyl-reduced LPS (B) of
A salmonicida The inset shows the ratio of
the native and amidated forms of LPS and
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 kV The high orifice voltage of Q-Star
(120 V) provided the environment to break a
polysaccharide into repeating units, giving
diagnostic fragments arising from the cleavage
of glycosidic bonds.
Fig 5 The proposed structure of the CPS and
O-chain polysaccharide of A salmonicida
strain 80204-1.
Trang 8In order to evaluate the sensitivity of the CE-ES-MS
approach, a series of purified A salmonicida LPS standards
containing between 100 lg and 5 lg, 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 was established that even at the
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 trisaccharide repeating units, and fragment 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 in order to confirm the CE-ES-MS
assignments, tandem MS was used for the precursor ions at
m/z663.3 and m/z 662.3, corresponding to the mass of one
trisaccharide repeating unit-containing species with
GalN-AcA and GalNGalN-AcAN, respectively As expected, two
fragment ions, at m/z 404.2 and m/z 259.2, respectively,
were observed, where a fragment ion at m/z 404.2
corres-ponded to the loss of Qui3NAlaNAc (258 Da), and a
fragment ion at m/z 259.2, corresponded to a protonated
Qui3NAlaNAc species
When direct CE-ES-MS analysis was carried out on the
in vivocultured cells of A salmonicida strain 80204-1, as
shown in Fig 6, the presence of m/z 662.3 could not be
unambiguously confirmed due to a high background which
also resulted in the loss of other characteristic fragment ions
at m/z 1067.4 and m/z 1325.5 However, CE-ES-MS/MS
analysis of the precursor ion at m/z 662.3 was consistent
with its previously determined composition of a
trisaccha-ride repeating unit consisting of Qui3NAlaNAc,
GalN-AcAN and QuiNAc and confirming its presence in the
in vivocultured cells Similarly, based on the CE-ES-MS/
MS analysis of the precursor ion at m/z 662.3, the same trisaccharide repeating unit could be also detected in 2.5% saline wash of the in vivo grown bacterial cells It is noteworthy that the fragment ion at m/z 663.3 correspond-ing to a trisaccharide-repeatcorrespond-ing unit containcorrespond-ing GalNAcA was not detected in the in vivo cultured cells, suggesting a complete amidation of GalNAcA
Discussion
In this study we have established the structure of the CPS and LPS O-chain polysaccharide of A salmonicida strain 80204-1 produced under in vitro growth conditions on TSA Both polysaccharides were shown by composition, methy-lation analysis, NMR and MS methods to be 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 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) anguillarumand 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 recently been re-examined and that the galacto configuration of the
Fig 6 CE-ES-MS and CE-ES-MS/MS ana-lysis (+ ion mode) of LPS from in vivo
A salmonicida strain 80204-1 cells Extracted mass spectra for LPS Extracted MS/MS spectra of precursor ion m/z 662.3 promoted using an orifice voltage of 120 V Other conditions were the same as in Fig 4.
Trang 92,3-diacetamido-2,3-dideoxy-hexuronic acid in both
struc-tures should be revised in favour of the gulo configuration
(Y A Knirel & A S Shashkov, personal communication)
In the present investigation, we have shown that the
structures of both CPS and O-chain polysaccharide were
distinct from the previously reported O-chain
polysacchar-ide of A salmonicida produced in TSB and consisting of
L-rhamnose,D-mannosamine andD-glucose [11] which was
also detected in the bacterial inoculum TSB culture used to
prepare the in vivo growth chambers Direct CE-ES-MS
analysis of the in vivo bacterial cells cultured within the
semipermeable growth chambers for 72 h has confirmed
the formation of a novel polysaccharide It is possible that
the use of dialysis tubing in the in vivo growth chambers
provides an environment more closely resembling that seen
in a systemic infection within salmon That environment
may be 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 lg of LPS in the sample
The present studies suggest that caution should be
exercised when in vitro-cultured cells are used for isolation
and structural analysis of bacterial polysaccharides as the
resultant structural information 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 be a powerful technique to
distinguish different glycoforms when applied to
lipopoly-saccharides from Neisseria meningitidis [28] However, in
this particular application we attempted to improve the
detection sensitivity by separating the CPS and LPS
from salts and other matrices associated with the sample
preparation
These findings suggest that additional virulence 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 Fisheries and Oceans Canada, Moncton, NB for the kind gift of
A salmonicida strains used in this study, Dr Jessica Boyd from the
Institute of Marine Biosciences, National Research Council of Canada
for providing A salmonicida stock cultures and the Dalhousie
University Aquatron for wetlab space We also thank Mrs Vandana
Chandan from the Institute of Biological Sciences, National Research
Council of Canada for excellent training in bacterial culture techniques.
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