jejuni NCTC 11168 revealed that the strain possessed a type II/III capsule locus found in other organisms such as E.. Sugar composition and methylation linkage analysis Sugar composition
Trang 1The structures of the lipooligosaccharide and capsule polysaccharide
Frank St Michael, Christine M Szymanski, Jianjun Li, Kenneth H Chan, Nam Huan Khieu,
Suzon Larocque, Warren W Wakarchuk, Jean-Robert Brisson and Mario A Monteiro
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada
Campylobacter jejuniinfections are one of the leading causes
of human gastroenteritis and are suspected of being a
pre-cursor to Guillain–Barre´ and Miller–Fisher syndromes
Recently, the complete genome sequence of C jejuni NCTC
11168 was described In this study, the molecular structure of
the lipooligosaccharide and capsular polysaccharide of
C jejuniNCTC 11168 was investigated The
lipooligosac-charide was shown to exhibit carbohydrate structures
anal-ogous to the GM1a and GM2 carbohydrate epitopes of
human gangliosides (shown below):
The high Mr capsule polysaccharide was composed of
b-D-Ribp, b-D-GalfNAc, a-D-GlcpA6(NGro), a uronic
acid amidated with 2-amino-2-deoxyglycerol at C-6, and
6-O-methyl-D-glycero-a-L-gluco-heptopyranose as a
side-branch (shown below):
The structural information presented here will aid in the identification and characterization of specific enzymes that are involved in the biosynthesis of these structures and may
lead to the discovery of potential therapeutic targets In addition, the correlation of carbohydrate structure with gene complement will aid in the elucidation of the role of these surface carbohydrates in C jejuni pathogenesis
Keywords: lipooligosaccharide; capsule; electron spray ionization mass spectrometry; high-resolution magic angle spinning NMR; heptose
In humans, Campylobacter jejuni infection often gives rise to enteritis and, in some regions, this Gram-negative bacterium surpasses Salmonella, Shigella and Escherichia as the primary cause of gastrointestinal disease [1,2] Moreover,
C jejuni infections have been linked to the more severe clinical outcomes caused by Guillain–Barre´ [3,4] and Miller–Fisher syndromes [5] The subsequent paralysis observed in Guillain-Barre´ and Miller–Fisher syndromes
is thought to be an autoimmune reaction due to molecular mimicry of gangliosides by C jejuni lipooligosaccharides (LOS) [6,7]
In the pioneering studies carried out by Aspinall and coworkers on the cell-surface carbohydrates from Campylobacterspecies, it was observed that insoluble gels from phenol-water extractions of bacterial cells yielded mainly low MrLOS, with core oligosaccharide linked to lipid A, and the aqueous phases from such extractions furnished high Mr glycans with extended polymers with
no attachment to lipid A as seen in the teichoic acid-like
P/PEtn
6 b-Gal-(1fi3)- b- D -GalNAc-(1fi4)-b- D -Gal-(1 fi3)-b- D -Gal-(1fi3)- L -a- D -Hep-(1fi3)- L -a- D -Hep-(1fi5)Kdo
a-Neu5Ac a- D -Gal b- D -Glc b- D -Glc
6-O-Me- D -a- L -glcHepp
1 fl 3 fi2)-b- D -Ribf-(1-5)-b- D -Galf NAc-(1-4)-a- D -GlcpA6(NGro)-(1fi
0
B
B
B
B
@
0 B B B B
@
1 C C C C A
Correspondence to J.-R Brisson, Institute for Biological Sciences,
National Research Council of Canada, Ottawa, Canada, K1A 0R6.
Fax: + 1 613 952 9092, Tel.: + 1 613 990 3244,
E-mail: jean-robert.brisson@nrc.ca; M Monteiro, Wyeth Vaccines
Research, 211 Bailey Road, West Henrietta, NY, 14586, USA.
Fax: + 1 585 273 751, Tel.: + 1 585 273 7667,
E-mail: Monteim@wyeth.com
Abbreviations: CE, capillary electrophoresis; ESI-MS, electron spray
ionization mass spectrometry; FAB, fast-atom bombardment;
HMBC, heteronuclear multiple bond coherence; HMQC,
heteronu-clear multiple quantum coherence; HR-MAS, high-resolution magic
angle spinning; HSQC, heteronuclear single quantum coherence;
KmR, kanamycin resistance; LOS, lipooligosaccharide; LPS,
lipo-polysaccharide; OS, oligosaccharide.
Dedication: The authors would like to dedicate this manuscript to
Professor Gerald Aspinall.
(Received 3 July 2002, revised 16 August 2002,
accepted 21 August 2002)
Trang 2polymer from C coli serotype HS:30, the repeating
disac-charide from C jejuni serotype HS:3, and the
poly(tetra-glycosylphosphates) from C lari [6]
Analysis of the genome sequence of C jejuni NCTC
11168 revealed that the strain possessed a type II/III capsule
locus found in other organisms such as E coli K1 and
Neisseria meningitidis group B [8,9] This led to the
realization that what was once believed to be high molecular
weight lipopolysaccharides (LPS) was actually capsule and
that capsule was the main serodeterminant of the Penner
typing scheme [10,11]
Campylobacter LOS and capsule are important in
adherence and invasion in vitro [10,12], colonization and
disease in vivo [10], molecular mimicry of gangliosides [6,7],
possible autoimmunity leading to Guillain–Barre´ and
Miller–Fisher syndromes [5,13], maintenance of cell surface
charge [10], antigenic and phase variation [10,12,14–17], and
serum resistance [10,15] Also, C jejuni NCTC 11168 was
recently sequenced [8] and serves as a reference strain for the
understanding of the genetics of this significant food-borne
pathogen We therefore elucidated the structures of these
major cell surface carbohydrates to make functional
analysis of the genes in the respective genetic loci possible
E X P E R I M E N T A L P R O C E D U R E S
Bacterial strains and plasmids
C jejuniNCTC 11168 (HS:2) was originally isolated from a
case of human enteritis [18] and later sequenced by Parkhill
et al [8] E coli DH10B (Invitrogen) was used as the host
for the cloning experiments Plasmid pPCR-Script Amp
(Stratagene) was used as the cloning vector
Media and growth conditions
C jejuni NCTC 11168 was routinely grown on Mueller
Hinton agar (Difco) under microaerophilic conditions at
37C E coli clones were grown on Luria S-gal agar
(Sigma) at 37C When appropriate, antibiotics were added
to the following final concentrations: kanamycin,
30 lgÆmL)1and ampicillin, 150 lgÆmL)1
Generation of LOS and capsule
Campylobacter biomass was harvested from overnight
liquid cultures by centrifugation Carbohydrates were
isolated by the hot water/phenol extraction of bacterial
cells [19] as a gel-like pellet upon ultracentrifugation of the
aqueous phase The LOS pellet was lyophilized, then
purified on a column of Bio-Gel P-2 (1 cm· 100 cm) with
water as eluent Some of the LOS preparation was then
treated with 1% acetic acid at 100Cfor 1 h with
subsequent removal of the insoluble lipid A by
centrifuga-tion (5000 g) to yield the core oligosaccharide (OS)
The supernatant from ultracentrifugation was purified on
Bio-Gel P-2 with water as eluent and lyophilized to obtain
the capsule polysaccharide (PS-1)
Sugar composition and methylation linkage analysis
Sugar composition analysis was performed by the alditol
acetate method [20] The hydrolysis was performed in 4M
trifluoroacetic acid for 4 h at 100Cfollowed by reduction with NaBD4in H2O overnight, then acetylated with acetic anhydride at 100Cfor 2 h using residual sodium acetate as catalyst The alditol acetate derivatives were characterized
by GLC-MS using a Hewlett-Packard chromatograph equipped with a 30-M DB-17 capillary column (180Cto
260Cat 3.5 CÆmin)1) MS was performed in the electron impact mode and recorded on a Varian Saturn II mass spectrometer Absolute configuration was assigned by characterization of the but-2-yl glycosides in GLC-MS [21] Methylation analysis was carried out by the NaOH/ dimethylsulfoxide/CH3I procedure [22] using GLC-MS in the electron impact mode to characterize the sugars as above A portion (1/4) of the methylated sample was used for fast-atom bombardment-mass spectrometry (FAB-MS)
in positive ion mode The Jeol JMS-AX505H mass spectrometer was used with a matrix of glycerol/thioglycerol
1 : 3 and 3 kV as the tip voltage
Smith degradation The polysaccharide sample ( 5 mg) was oxidized with
40 mM sodium metaperiodate in 0.1M sodium acetate at
4Cfor 72 h [23] The product was isolated on a Bio-Gel P-2 as per above then reduced with NaBD4and acidified with cation exchange resin (J T Baker) The product was then hydrolyzed with 1Mtrifluoroacetic acid at 45Cfor
1 h, reduced with NaBD4and re-acidified Then, finally the sample was fractionated on a Bio-Gel P-2 column and fractions analyzed
CE-ESI-MS and CE-ESI-MS/MS
A crystal Model 310 capillary electrophoresis (CE) instrument (AYI Unicam) was coupled to an API 3000 mass spectrometer (Perkin-Elmer/Sciex) via a microIon-spray interface A sheath solution (isopropanol/methanol
2 : 1) was delivered at a flow rate of 1 lLÆmin)1to a low dead volume tee (250 lm internal diameter, Chromato-graphic Specialties) All aqueous solutions were filtered through a 0.45-lm filter (Millipore) before use An electrospray stainless steel needle (27 gauge) 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 about 90-cm length bare fused-silica capillary using 10 mMammonium acetate/ ammonium hydroxide in deionized water, pH 9.0, con-taining 5% methanol A voltage of 20 kV was typically applied at the injection The outlet of the capillary was tapered to c 15 lm internal diameter using a laser puller (Sutter Instruments) Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z)1unit in full-mass scan mode The MS/MS data were acquired with dwell times of 1.0 ms per step of 1 m/z)1 unit Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell, were mass-analyzed by scanning the third quadrupole
Construction and characterization
of insertional mutants For construction of the Cj1428c mutant, genes Cj1427c to Cj1428c were PCR amplified from C jejuni NCTC 11168
Trang 3with the following primer pair: Cj1427cF918 (5¢-AA
CTTTCATCATTTTAAACGCTCTT-3¢) and fclR51
(5¢-TACAGCATTGGTAGAAAACTTACAA-3¢) For
construction of the kpsM mutant, gene Cj1448c was PCR
amplified with: kpsMF771 (5¢- TACCGCCGTTAAAGCT
TGTCTATTA-3¢) and kpsMR73B (5¢- TATATATGGGT
AGTTGGGGAGCCTA-3¢) For construction of the
Cj1439c mutant, gene Cj1439c was PCR amplified with:
glfF1081 (5¢-TTTTACAAAATAATAATGCCGATCT-3¢)
and glfR6 (5¢-TGATTATTTAATTGTTGGTTCTGG
A-3¢) The PCR products were ligated to pPCR-Script
Amp according to the manufacturer’s instructions A
blunt-ended kanamycin resistance (KmR) cassette from pILL600
[24] was inserted into the filled-in BglII restriction site of
Cj1428c to create pCSc28, into the NheI restriction site
of kpsM to create pCSc48, and into the BsaBI restriction
site of Cj1439c to create pCSc39 The orientation of the
KmR cassette was determined by sequencing with the
ckanB primer (5¢-CCTGGGTTTCAAGCATTAG-3¢)
using terminator chemistry and AmpliTaq DNA
polym-erase FS cycle sequencing kits (Perkin Elmer-Applied
Biosystems) and analyzed on an Applied Biosystems 373
DNA sequencer The mutated plasmid DNA was used for
electroporation into C jejuni NCTC 11168 [25] and KmR
transformants were characterized by PCR to confirm that
the incoming plasmid DNA had integrated by a double
cross-over event
Reverse transcriptase-polymerase chain reaction
It has previously been shown that gene insertion of the
CampylobacterKmR cassette in a nonpolar orientation has
no effect on transcription of downstream genes [26]
Sequencing results confirmed that the KmR insertion in
both kpsM and Cj1439c was in the nonpolar orientation
However, the KmR cassette inserted in a polar orientation
into Cj1428c so, we wanted to determine whether Cj1428c
disruption had a polar affect on downstream genes RNA
was isolated from C jejuni wild type and the Cj1428c
mutant using the RNeasy mini kit (Qiagen) RT-PCR
reactions, along with controls, were performed using the
OneStep RT-PCR kit (Qiagen) First-strand synthesis was
carried out as described by the manufacturer at 50Cfor
30 min PCR conditions were 95Cfor 15 min followed by
30 cycles at 94Cfor 30 s, 59 Cfor 4 min, and 72 Cfor
30 s followed by a final annealing at 72Cfor 10 min using
primers: Cj1427cF918 (5¢-AACTTTCATCATTTTAAAC
GCTCTT-3¢) and Cj1427cR10 (5¢-AAAGTTTTAATTAC
AGGTGGTGCAG-3¢)
Deoxycholate-PAGE analysis and silver staining
of polysaccharides
Proteinase K-treated whole cells of C jejuni were
prepared according to the method of Salloway et al
[27] based on the original method of Hitchcock and
Brown [28] The samples were separated on 16.5%
deoxycholate-PAGE [29] After electrophoresis, the gels
were silver stained according to the method of Tsai and
Frasch [30] However, gels were fixed for 2–5 h rather
than overnight to prevent elution of the high molecular
weight polysaccharides [31] In addition, we have recently
improved the silver staining procedure by visualizing the
carbohydrates with commercially available developer (Bio-Rad [32])
Nuclear magnetic resonance NMR experiments were acquired on Varian Inova 600,
500 and 400 MHz spectrometers using a 5-mm triple resonance probe with the 1H coil nearest to the sample and with a Z gradient coil All measurements were made
at 25Con 2–5 mg of sample dissolved in 0.6 mL of
D2O, pH 6–7 Experiments in 90% H2O were carried out at pH 4–5 The methyl resonance of acetone was used as an internal reference at 2.225 p.p.m for 1H spectra and 31.07 p.p.m for 13Cspectra Standard homo and heteronuclear correlated 2D techniques were used for general assignments: COSY, TOCSY, NOESY,
Selective 1D experiments were performed for the determination of accurate coupling constants and NOEs and to perform the 1D analog of a 3D-TOC SY-NOESY experiment [33] High resolution magic angle spinning (HR-MAS) experiments were performed using
a gradient 4 mm indirect detection nano-NMR probe (Varian) with a broadband decoupling coil Proton spectra of killed cell pellets were acquired as described previously [34]
Molecular modeling The conformational analysis for trisaccharide BCD of PS-1 was performed using the Metropolis Monte-Carlo method
as previously described [35] The PFOS potential was used [36] Residue B was modeled as a glucuronic acid Minim-ized coordinates for the monosaccharides were obtained using MM3(92) available from the Quantum Chemistry Program Exchange The minimum energy conformation for each disaccharide was used as the starting conformation for the trisaccharide The (O5–C5–C6–O6) torsion angle was restricted to the)60 conformer for theDLform of residueD and to +60 for theLDform For the trisaccharide, 5· 103 macro moves were used with a step length of 10 for the glycosidic linkage and pendant groups and a temperature of
103K resulting in an acceptance ratio of 0.5 Distances were extracted from the saved coordinates at each macro move The molecular model for the trisaccharides were generated using the minimum energy conformer Molecular drawings were performed using Schakal97 from E Keeler, University
of Freiburg, Germany
Vacuum MD simulation were performed with the DISCOVER-3 program from Accelrys Inc (MSI) using the AMBER FORCEFIELDversion 1.0–1.6, with Homans’ param-eters applicable to saccharides [37], on a SGI Indigo 2 Solid Impact R10000 195 MHz As before, the (O5–C5–C6–O6) torsion angle for residue D was restrained The initial structure was subjected to a 300-step energy minimization (BFGS method), followed by 50 ps dynamics simulation at
298 K Initial velocities were generated from a Maxwell– Boltzmann distribution, and the temperature was controlled
by the direct velocity scaling method The Verlet algorithm
of integrating the Newton’s equations of motion was applied with 1 fs timestep for the simulation where a distance-dependent dielectric of the form er (r¼ the distance between atoms) was used Distances were extracted
Trang 4from a trajectory file of 5000 frames stored after each MD
run
R E S U L T S
Structural determination of the LOS
The alditol acetate derivatives [20] of D-glucose (D-Glc),
D-galactose (D-Gal), N-acetyl-D-galactosamine (D-GalNAc)
andL-glycero-D-manno-heptose (LD-Hep), in the respective
ratios of 3 : 2.3 : 1 : 1.8, were detected in both the lipid
A-free core OS and intact LOS, by GLC-MS The absolute
configurations of the sugars mentioned above were deduced
by the identification of but-2-yl chiral glycosides in
GLC-MS [21] Sugar linkage analysis (Table 1), by the
methyla-tion procedure [22], on the LOS revealed the following sugar
linkage types: two units of terminalD-Glc, one unit of each
terminalD-Gal, 2,3-substitutedD-Gal and 3,4-disubstituted
D-Gal, traces of 4-substitutedD-Gal, one unit of terminal
D-GalNAc, trace amounts of 3-substituted D-GalNAc,
one unit of 2,3-disubstituted LD-Hep, and trace amounts
of 3,4-disubstitutedLD-Hep A parallel linkage analysis on
the liberated core OS, after removal of lipid A with 1% acetic
acid, afforded the same sugar linkage types, but in addition it
showed a significant decrease of 3,4-disubstitutedD-Gal and
a greater amount of 4-substitutedD-Gal (Table 1)
To gain a quick insight into the overall composition of the
LOS, a series of ESI-MS experiments were performed on
the core OS The ESI-MS spectrum (Fig 1a) of the core OS
showed a heterogeneous mixture (Table 2) with the
pres-ence at the reducing-end of the anhydro form of
3-deoxy-manno-octolusonic acid (Kdo) as a distinct marker The
primary molecular ion at m/z 1759 [)18 (H2O)]
correspon-ded to a composition of Hex5, Hep2, GalNAc, Kdo and
2-amino ethyl phosphate (PEtn), and ion m/z 1716 ()18)
belonged to a composition of Hex5, Hep2, GalNAc, Kdo
and phosphate (P) Trace amounts of the phosphate-free
core OS (Hex5, Hep2, GalNAc, Kdo) at m/z 1636 ()18)
was also observed, along with small amounts of m/z of 1877
and 1921, which corresponded to the addition of an extra
hexose to both phosphorylated core OSs For both
molecular ions, a significant ion at m/z 2007 ()18) (Hex5, Hep2, GalNAc, Kdo, Neu5Ac, P) and m/z 2050 ()18) (Hex5, Hep2, GalNAc, Kdo, Neu5Ac, PEtn) pointed towards the presence of sialic acid (Neu5Ac) in the core
OS Indeed, ESI-MS on core OS preparations that were obtained by a harsher treatment with 5% acetic acid, to intentionally remove any acid labile Neu5Ac, yielded the same primary ions as discussed above, but no ions containing sialic acid Taking into account the previous observed variation between 3,4-disubstituted Gal and 3-substituted Gal in the core OS, before and after mild acid treatment, and the detection of the acid sensitive sialic acid
in ESI-MS, suggested that Neu5Ac may be attached at O-3
of the 3,4-disubstituted Gal
The CE-MS/MS spectra for the components having a total mass of m/z 2050 or precursor ions at m/z 1026 (doubly protonated) are presented in Fig 1b The fragment ions observed at m/z 1848 and 1760 clearly indicated that one HexNAc residue and one Neu5Ac residue were present as terminal units As shown in the Fig 1b, the fragment ion of m/z1848, arising from the loss of HexNAc, subsequently loses one hexose (m/z 1686), one Neu5Ac (m/z 1394.5), three hexoses (m/z 1232, 1070, 908) and finally one heptose residue (m/z 716) The fragment ion at m/z 366 suggested that the HexNAc was attached to a Hex unit, and the fragment ion at m/z 454 was indicative a Hex-Neu5Ac disaccharide Moreover, fragment ion m/z 554 suggested the existence of PEtn-Hep-Kdo moiety It was also observed that a minor glycoform, with an extra Hex (composition of Hex6HexNAc1PEtn1Hep2Kdo1) was present in the LOS
of C jejuni NCTC 11168 The tandem mass spectrum of ion m/z1005, which corresponded to the morpholine adduct of the LOS, is presented in Fig 1c The doubly charged ion yielded a fragment ion at m/z 1922 when the morpholine ion was cleaved off The dissociation of the core oligosaccharide gave rise to consecutive losses of Hex, HexNAc, and five hexoses In contrast with Fig 1(b), in which the ion m/z 292 corresponded to a protonated Neu5Ac, no sialic acid fragment ion was found in this glycoform
For the core structural features of oligosaccharide from
C jejuni NCTC 11168, we have shown evidence for the proposed structure as indicated in the inset of Fig 1b Solid information regarding the sequences of sugar units was obtained by FAB-MS of the methylated core OS derivative Figure 2 and Table 3 shows a series of A-type primary glycosyl oxonium ions, and secondary ions, of defined compositions at m/z 260fi228 (GlcNAc)+, m/z 376fi344 (Neu5Ac)+, m/z 464 (Gal, GalNAc), m/z 826 (GalNAc, Neu5Ac, Gal)+, m/z 872 (GalNAc,Gal3)+, m/z
1029 (Hex2, GalNAc, Neu5Ac)+, m/z 1120 (GalNAc, Hex3, Hep)+, m/z 1233 (GalNAc, Hex3, Neu5Ac)+, m/z 1324 (GalNAc, Hex4, Hep)+, m/z 1407 (Hex4, Hep2, P)+, m/z
1477 (Hex4, Hep, Hep, PEtn)+, m/z 1685 (GalNAc, Hex4, Neu5Ac, Hep2)+, m/z 1857 (GalNAc, Hex5, Hep2, P)+and m/z1928 (GalNAc, Hex5, Hep2, PEtn]+ A double cleavage ion containing a phosphate moiety was also see at m/z 547 (Hep, Glc, P)+
Combining the FAB-MS sequence data (Fig 2, Table 3) with the information obtained from the linkage analysis (Table 1) and from the selective ESI-MS experiments (Fig 1, Table 2) The following provisional structural arrangement for the core OS region can be proposed (Hex¼ Glc or Gal):
Table 1 Methylation linkage analysis of C jejuni NCTC 11168 intact
LOS, core OS and Smith degradation products.
Linkage type LOS OS
Smith degradation product
fi4)-Gal-(1fi Traces < 1 Traces
fi2,3)-Gal-(1fi 1 1
fi3,4)-Gal-(1fi 1 < 1 Traces
GalNAc-(1fi 1 1 Traces
fi3)-GalNAc-(1fi Traces Traces
fi2,3)-Hep-(1fi 1 1
– fi3,4)-Hep (1fi Traces Traces
fi5)-3d-Hexitol a 0.5
fi5)-3d-Hexitol b 0.5
a,b
Two isomeric forms of 3-deoxy-1,1,2,6-tetra-2
H-5-O-acetyl-1,2,4,6-tetra-O-methyl-hexitol (from 5-substituted Kdo).
Trang 5A Smith degradation [23] was strategically performed on
the core OS to disentangled the linkages at the branch
points Prior to periodate oxidation, the core OS was
reduced with NaBD4for the incorporation of deuterium at
the Kdo terminus so that this unit could be detected in the
final product Periodate oxidation of the reduced core OS
was followed by reduction with NaBD4, mild acid
hydro-lysis, and a final reduction with NaBD4 Sugar linkage
analysis of the final product (Table 1) showed the presence
of terminal Gal (from 3,4-substituted Gal), 3-substituted
Gal (from 2,3-substituted Gal), 3-substituted Man-O-6-2H
(from 2,3- and 3,4-substituted LD-Hep units) and two
isomeric units of 3-deoxy-1,1,2,6-tetra-2 H-5-O-acetyl-1,2,4,6-tetra-O-methyl-hexitol (from 5-substituted Kdo), in the approximate ratios of 3 : 1: 3 : 0.5 : 0.5 There were also traces of 4- and 3,4-substituted Gal and terminal GalNAc; these two derivatives originated from an extended molecule that contained a hexose at the nonreducing terminus of the core {Hex-(1fi4)-GalNAc-(1fi4)[Neu5Ac-(1fi3)]-Gal… inner core} The isomeric hexitol derivatives were recognized as originating from a modified 5-linked Kdo termini as seen in all C jejuni strains The backbone Gal and
LD-Hep units are thus joined by 1fi3 linkages and the inner most LD-Hep is linked to O-5 of Kdo The FAB-MS
Fig 1 Electron spray ionization-mass
spectr-ometry C jejuni NCTC 11168 core OS
showing a heterogeneous mixture (a) CE-MS/
MS (+ ion mode, produces ions of m/z 1026)
analysis of C jejuni NCTC 11168 core OS (b).
CE-MS/MS (+ ion mode, produces ions of
m/z 1005) analysis of LOS from C jejuni
NCTC 11168 core OS (c).
P/PEtn fl [ D -Hex-(1fi3)]±- D -GalNAc-(1fi3 or 4)- D -Gal-(1fi2 or 3)- D -Gal-(1fi2 or 3)- LD -Hep-(1fi3 or 4)- LD -Hep-(1fiKdo
3 or 4 2 or 3 2 or 3 3 or 4
Neu5Ac D -Hex D -Hex D -Hex
Trang 6spectrum (Table 3) of the methylated final product from the
Smith degradation yielded some limited, but corroborating,
sequence data (*¼ deuterium) stemming from the terminus
showing m/z 219 (Gal)+, m/z 424 [Gal-(1fi3)-Man*]+, and
m/z289 (Neu5Ac*)+, m/z 493 [Neu5Ac*(1fi3)-Gal]*, and
m/z 902 [Neu5Ac*(2fi3)-Gal-(1fi3)-Gal-(1fi3)-Man*]+
Two final products were thus recovered from the Smith
degradation (shown below), one was terminated by a GM2
structure, and the major product was a linear
Gal-Man-Man-3dhex (shown below) backbone:
Two unresolved anomeric resonances, characteristic of
sugars with the mannose configuration, were observed at d
5.207 and d 5.07 in the 1H nuclear magnetic resonance
spectrum of the Smith degradation product The same
spectrum also showed one b anomeric resonance at 4.55
(J1,27.2 Hz) The1H NMR data just described indicated
that the Man (LD-Hep) units possessed an a anomeric
configuration, whereas the 2,3-disubstitutedD-Gal had a b
anomeric configuration Therefore, at this time, the
follow-ing structure for the core OS region, where Hex represents
Glc or Gal, was proposed:
The sugar linkage analysis (Table 1) performed on the
core OS suggested the presence of slightly more than one
unit of terminal Gal and two units of terminal Glc Given
the fact that the hexose at the nonreducing terminus was
only present in trace amounts, as observed by linkage
analysis (traces of 3-substituted GalNAc), ESI-MS and FAB-MS, the three side-branches hexoses could be assigned to two units of Glc and one unit of Gal The nonstoichiometric hexose present in trace amounts that is connected to the O-3 position of GalNAc was a Gal residue
The1H NMR spectrum, in combination with a1H–1H TOCSY experiment, of the core OS yielded three a anomeric protons, at d 5.67 (J1,23 Hz) that was assigned to
an a-D-Gal residue [H-2 d 3.79 (J2,39.5 Hz) and H-3 d 3.93 (J3,43 Hz)], and at d 5.40 (unresolved doublet) [H-2 d 4.39 (J2,3 2 Hz) and H-3 d 4.28 (J3,4 3 Hz)], and at 5.10 (unresolved doublet) [H-2 d 4.17 (J2,3 2 Hz)], typical of a-D-Man configurations and were thus assigned to the
L-a-D-Hep residues, as were observed in the Smith degradation1H NMR product described above All other anomeric resonances detected possessed b anomeric con-figuration and could be seen at d 4.99 (J1,2)7 Hz), d 4.88 (J1,2)7 Hz), d 4.69 (J1,2)7 Hz), d 4.66 (J1,2)7 Hz) and d 4.62 (J1,2)7 Hz) To situate the side-branch hexoses, a 2D
1H–1H NOESY experiment was performed and conclusive evidence, an inter-NOE between H-1 (d 5.67) of the a-Gal and H-2 (d 3.98) of the residue with the b anomeric H-1 (d 4.99), was obtained that placed the sole a-D-Gal side-branch at O-2 of the unit with the anomeric at d 4.99, which has to belong to the sole 2-substituted unit, that being the 2,3-disubstituted Gal in the backbone The other side-branch hexoses, two Glc units, must then have the b anomeric configuration and be attached to
the L-a-D-Hep residues (b-D-Glc-(1fi2)-L-a-D-Hep) and (b-D-Glc-(1fi4)-L-a-D-Hep)
The GM2 and GM1a ganglioside mimics in C jejuni NCTC 11168 LOSs were covalently attached to the inner core region (Fig 3) composed of basal core OS units, Gal,
LD-Hep and Glc The GM2 and GM1a epitopes completed
a core OS similar to that present in C jejuni serogroup HS:1 [6] In addition, the innermost LD-Hep was phos-phorylated by a monoester phosphate or by a 2-amino ethyl phosphate
Structure of the capsule polysaccharide The polysaccharide, obtained from the aqueous phase after ultracentrifugation, was purified on a Bio-Gel P-2 (PS-1) Alditol acetate analysis revealed the presence of -Glc,
Table 2 Negative ion ESI-MS data and proposed compositions for
C jejuni NCTC 11168 core OS and de-O-acylated LOS (masses include
the addition of water)a.
Core OS
Observed molecular
mass (Da)
Proposed structure
1635 ( )18) HexNAcÆHex 5 ÆHep 2 ÆKdo
1714 ( )18) HexNAcÆHex 5 ÆHep 2 ÆPÆKdo
1758 ( )18) HexNAcÆHex 5 ÆHep 2 ÆPEtnÆKdo
1876 HexNAcÆHex 6 ÆHep 2 ÆPÆKdo
1920 HexNAcÆHex 6 ÆHep 2 ÆPEtnÆKdo
2005 ( )18) Neu5AcÆHexNAcÆHex 5 ÆHep 2 ÆPÆKdo
2049 ( )18) Neu5AcÆHexNAcÆHex 5 ÆHep 2 ÆPEtnÆKdo
a Residues used and their molecular mass: Neu5Ac, 291; HexNAc,
203; Hex, 162; Hep, 192; PEtn, 123; P, 79; Kdo, 220.
P/PEtn fl [ D -Hex-(1fi3)]±- D -GalNAc-(1fi4)- D -Gal-(1fi3)-b- D -Gal-(1fi3)- L -a- D -Hep-(1fi3)-L-a- D -Hep-(1fi5)-Kdo
Neu5Ac D -Hex D -Hex D -Hex
Major Smith degradation product
[ D -GalNAc-(1fi4)- D -Gal-(1fi3)]±- D -Gal-(1fi3)- D -Man*-(1fi3)- D -Man*-(1fi)-3-d-hexitol****
3
› 2 [Neu5Ac*]±
Trang 7D-Gal,LD-Hep (from the core region) and D-GlcNAc as
minor components, and as major unitsD-ribose (D-Rib),
6-O-methyl-heptose (6-O-Me-Hep), and N-acetyl-D
-gal-actosamine (D-GalNAc) From the alditol acetate analysis
it was observed that PS-1 was slightly contaminated with
LOS, additional efforts at purification were not successful
The methylation linkage analysis revealed the presence
of 2-substituted D-Rib, 4-substituted D-GalNAc, and a
terminal heptose unit No substituted heptose was detected,
and thus the 6-O-Me-Hep was present as a side chain
residue
For further characterization of the structure of PS-1, the
sample was acid hydrolyzed under mild conditions (1 M
HCl, 100Cfor 5 min) and the resultant hydrolysate was
purified on a Bio-Gel P-2 column The sample was then
analyzed by CE-ESI-MS (Fig 4a) and gave rise to two
components having a mass of 791 as the major product and
a minor mass of 762 The MS/MS spectra of m/z 791
(Fig 4b) revealed fragments m/z 588 and 585 This showed
the loss of either 6-O-Me-Hep or GalNAc, respectively; thus
illustrating that they were terminal units in this OS from
acid hydrolysis of PS-1 This finding was consistent with
the previous observation in the linkage analysis that the
6-O-Me-Hep was a terminal unit The fragment ion at 382
arose from the loss of both the 6-O-Me-Hep and GalNAc,
which then lead to the loss of Rib with a final mass of
250 Da The MS/MS spectra of m/z 762 (Fig 4c), as
with m/z 791, showed the loss GalNAc (m/z 558) and
6-O-Me-Hep (m/z 555) as terminal residues It can also be
observed, as in the previous example, that after the loss of
the 6-O-Me-Hep and the GalNAc leading to fragment ion m/z352, Rib is also lost, which furnished a final mass of
220 Da Therefore, from this CE-MS/MS analysis it was suspected that there might be two polysaccharide chains present or that one of the components in the capsule can be modified
The1H NMR spectrum of PS-1 (Fig 5a,b) revealed the presence of four anomerics COSY, NOESY, and TOCSY NMR experiments were performed on PS-1, but the heterogeneity lead to broad lines, making interpretation difficult This lead to the use of HR-MAS NMR to examine capsular polysaccharide resonances on intact Campylobacter cells without the need for extensive growth and purification [34] HR-MAS spectra of wild-type and capsule mutants were used for the initial screening and selection of a mutant that lacked the 6-O-Me-Hep, as this sugar was suspected to
be a side chain The disappearance of one anomeric and the loss of the OMe resonance at 3.56 p.p.m (Fig 5c,d) were used to ascertain this Once an appropriate mutant was generated (Cj1428c mutant; see below), its polysaccharide was purified (the absence of the 6-O-Me-Hep was also verified by alditol acetate analysis, results not shown) The polysaccharide (isolated as described above) of the Cj1428c mutant was denoted as PS-2 (Fig 6) The proton spectrum
of PS-2 was more homogeneous and had sharper lines than the proton spectrum of PS-1 As the spectrum of PS-2 was less complex than the spectrum of the native PS-1, its structural determination was first undertaken
NMR methods, as outlined before [33,38,39], were used for the structural determination of polysaccharides 1 and
Fig 2 FAB-MS spectra of the methylated
C jejuni NCTC 11168 core OS.
Trang 8Table 3 Interpretation of m/z ions in the FAB-MS spectrumof the methylated core OS fromC jejuni NCTC 11168.
Primary m/z ion Secondary m/z ion Double cleavage m/z ion Proposed structure
547 Glc-(1fi4)-Hep +
› P
3
› 2 Neu5Ac
3
› 2 Neu5Ac
3
› 2 Neu5Ac
1120 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep +
2
› 1 Gal
Neu5Ac Gal
1324 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep +
Gal Glc
fl Gal-(1fi3)-Hep-(1fi3)-Hep +
Gal Glc Glc
fl Gal-(1fi3)-Hep-(1fi3)-Hep +
GalGlc Glc
1685 GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep +
Neu5Ac Gal Glc
Trang 92 obtained from C jejuni 1D selective NMR methods
were also used to characterize individual components [33]
1D-TOCSY experiments were used to detect the coupled
spin systems and spin simulation was used to obtain
accurate coupling constants (Figs 7a–d) HMQCwas used
to assign CH, CH2and CH3carbon resonances (Fig 7f)
The assignments were verified by means of an
HMQC-TOCSY experiment HMBC was used to locate the C¼O
resonances (Fig 7g) A correlation was also observed
between C-7C and H-8C for the NAc group of residue C (results not shown) Location of nitrogen-bearing groups was carried out by performing experiments in 90% H2O in order to detect the NH resonances (Fig 7i) A COSY experiment (results not shown) was used to detect the NH-C-H correlation and assign the NH resonances The NOESY experiment was used to detect NOEs between the pendant groups and the ring protons Finally the HMBCexperiment was used to detect the multiple bond correlation between the C¼O and NH resonances (Fig 7i)
Table 3 (Continued).
fl GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep-(1fi3)-Hep +
Neu5Ac Gal Glc Glc
fl GalNAc-(1fi4)-Gal-(1fi3)-Gal-(1fi3)-Hep-(1fi3)-Hep +
Neu5Ac Gal Glc Glc Atomic mass units of the residues discussed above: Glc/Gal GalNAc Hep Neu5Ac P PEtn
Fig 3 The complete structure of C jejuni
NCTC 11168 LOS.
Fig 4 CE-ESI-MS and CE-MS/MS analysis CE-ESI-MS of PS-1
after acid hydrolysis (1 M HCl, 100 Cfor 5 min) and Bio-Gel P-2
purification (a), C E-MS/MS of m/z 791 (b), CE-MS/MS of m/z 762 (c).
Fig 5 Proton spectra of C jejuni whole cells and isolated polysaccha-rides HR-MAS spectrum of C jejuni whole cells (a) and its isolated polysaccharide (b) HR-MAS spectrum of C jejuni Cj1428c mutant whole cells (c) and its isolated polysaccharide (d) The anomeric resonances are labeled according to the structures shown in Fig 6.
Trang 10As the absolute configuration of the sugars was known from the chemical analysis, from a comparison of chemical shifts and coupling constants with those of monosaccharides, residue A was assigned as b-D-Ribf, residue B as the amide
of a-D-GlcpA with -NH-CH2-CH2OH at C-6, and residue C
as b-D-GalfNAc The sequence of sugars was established by
an HMBC experiment (Fig 7h) The (H-1 A, C-5C) (H-1C, C-4B) and (H-1B, C-2 A) HMBC correlations established the (-A-C-B-)npolymeric sequence (Fig 6) The NMR data are shown in Table 4
The linkage analysis of the Cj1428c mutant, PS-2, by the methylation method [22] revealed the same 2-substituted Rib and 4-substituted GalpNAc, which in this case, as observed by NMR, is a 5-substituted GalfNAc As expected, this PS-2 lacked the terminal 6-O-Me-Hep Confirmation of the repeat established by NMR was observed in the
FAB-MS and MALDI-FAB-MS (Fig 8) of the methylated PS-2 The MALDI-MS spectra shows a series of A-type primary glycosyl oxonium ions and secondary ions of defined compositions at m/z 260fi228 (GalNAc)+, m/z 420 (Gal-NAc, Rib)+, m/z 695 (GalNAc, Rib, GlcA), m/z 941 (GalNAc2, Rib, GlcA)+, m/z 1101 (GalNAc2, Rib2, GlcA)+, m/z 1620 (GalNAc3, Rib2, GlcA2)+, m/z 1781
Fig 6 Structure of polysaccharides PS-1 and PS-2 from C jejuni, and
labeling of the residues and atoms Residue A is b- D -Ribf, residue B is
the amide of a- D -GlcpA with ethanolamine at C-6 for PS-2 and with
2-amino-2-deoxyglycerol at C-6 for PS-1, residue C is b- D -GalfNAc,
and residue D is D -glycero-a- L -gluco-heptopyranose.
Fig 7 NMR spectrumof PS-2 Spin simula-ted spectra for residue A (a), residue B (b), residue C(c) and the substituent at C-6 of residue B (d), along with the resolution enhanced proton spectrum (e) In (f) is the HMQCspectrum of the ring protons In (g)
is the HMBCspectrum showing the C ¼O region for assignment of C-6B and C-7C The HMBCspectrum in (h) shows the intergly-cosidic3J(C,H) correlations In (i) the proton spectrum, NOESY and HMBCcorrelations for the NH resonances obtained in 90% H 2 O are shown.