Structures of two O-chain polysaccharides of Citrobacter gillenii 09a,9b lipopolysaccharide A new homopolymer of 4-amino-4,6-dideoxy-p-mannose perosamine Tomasz Lipinski’, George V.. Z
Trang 1Structures of two O-chain polysaccharides of Citrobacter gillenii 09a,9b lipopolysaccharide
A new homopolymer of 4-amino-4,6-dideoxy-p-mannose (perosamine)
Tomasz Lipinski’, George V Zatonsky”, Nina A Kocharova’, Michel Jaquinod?, Eric Forest?,
Alexander S Shashkov’, Andrzej Gamian' and Yuriy A Knirel”
'L Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; *N D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation; 7>CNRS and CEA, Institut de Biologie
Structurale, LSMP, Grenoble, France
Mild acid degradation of the lipopolysaccharide of Citro-
bacter gillenii 09a,9b released a polysaccharide (PS), which
was found to consist of a single monosaccharide, 4-
acetamido-4,6-dideoxy-D-mannose (D-Rha4NAc, N-acetyl-
D-perosamine) PS was studied by methylation analysis and
"H-NMR and "C-NMR spectroscopy, using two-dimen-
sional 'H,'H COSY, TOCSY, NOESY, and H-detected
'H,'°C heteronuclear correlation experiments It was found
that PS includes two structurally different polysaccharides:
an «1 — 2-linked homopolymer of N-acetyl-p-perosamine
[ > 2)-o-p-Rhap4NAc-(1 >, PS2] and a polysaccharide
composed of tetrasaccharide repeating units (PS1) with
the following structure: — 3)-o-p-Rhap4NAc-(1 > 2)-a-
b-Rhap4NAc-(1 — 2)-o-p-Rhap4NAc-(1 — 3)-a-p-Rhap4
N Ac2Ac-(1 > where the degree of O-acetylation of a
3-substituted Rha4NAc residue at position 2 is ~ 70%
PS could be fractionated into PSI and PS2 by gel-perme- ation chromatography on TSK HW-50S Matrix-assisted laser desorption ionization MS data indicate sequential chain elongation of both PS1 and PS2 by a single sugar unit, with O-acetylation in PSI beginning at a certain chain length Anti-(C gillenii 09a,9b) serum reacted with PS1 in double immunodiffusion and immunoblotting, whereas neither PS2 nor the lipopolysaccharide of Vibrio cholerae O1 with a structurally related O-chain polysaccharide were reactive
bacter gillenii; lipopolysaccharide; O-antigen; polysaccharide
structure
Strains of genus Citrobacter are inhabitants of the intestinal
tract and, accordingly, are present in sewage, surface waters,
and food contaminated with faecal material Outbreaks of
febrile gastroenteritis associated with Citrobacter have been
described Citrobacter strains may cause opportunistic
infections, including urinary and respiratory tract infections,
especially in the immunocompromised host, and are also
associated with meningitis, brain abscesses, and neonatal
sepsis [1,2] Currently, strains of the genus Citrobacter are
classified into 11 species [3] and 43 O-serogroups [1,4]
Serological heterogeneity of Citrobacter strains is defined by
the diversity in structures of the cell-surface lipopolysac-
charide (LPS) [1,5] With the aim of creating a molecular
basis for classification of strains and substantiating their
serological cross-reactivity, structures of the O-chain poly-
saccharides of LPS (O-antigens) of more than 20 serologi-
Correspondence to A Gamian, L Hirszfeld Institute of Immunology
and Experimental Therapy, Polish Academy of Sciences, Weigla 12,
53-114 Wroclaw, Poland Fax: + 48 71 3732587,
Tel.: + 48 71 3732316, E-mail: gamian@immuno.iitd.pan.wroc.pl
Abbreviations: HSQC, heteronuclear single-quantum coherence;
MALDI, matrix-assisted laser desorption ionization; LPS, lipopoly-
saccharide; PS, O-chain polysaccharide; Rha4NAc, 4-acetamido-4,6-
dideoxymannose
(Received 22 June 2001, revised 22 October 2001, accepted 23 October
2001)
cally different Citrobacter strains have been established
[6-8] Now we report structural studies of LPS from
C gillenii O9a,9b, which is distinguished by the presence of two structurally different polysaccharide chains Strains of this serogroup are often isolated from patients [1]
MATERIALS AND METHODS Bacterial strain, isolation and degradation of LPS Citrobacter gillenii O9a,9b:48 (strain PCM 1537) came originally from the Czech National Collection of Type Cultures, Prague (THE Be 65/57, Bonn 16824 [1,5,9]) and was obtained from the collection of the Institute of Immunology and Experimental Therapy Bacteria were cultivated in Davis broth supplemented with casein hydro- lysate and yeast extract (Difco) with aeration at 37 °C for
24 h; they were then harvested and freeze-dried LPS was isolated by phenol/water extraction and purified by ultra- centrifugation [10] The yield of LPS was 3.2% of dry bacterial mass
A portion of LPS (200 mg) was heated with 1% acetic acid (20 mL) for 3h at 100 °C, and the carbohydrate- containing supernatant was fractionated on a column (1.6 x 100 cm) of Bio-Gel P4 (—400 mesh) in 0.05 M aqueous pyridinium acetate buffer, pH 5.6, at a flow rate
of 4mL‘h The yield of polysaccharide material was
34 mg Alternatively, carbohydrate material from another
Trang 2Chemical methods
O-deacetylation of PS (30 mg) was carried out with aqueous
12% ammonia at room temperature overnight followed by
gel-permeation chromatography on a column (1.6 x 80 cm)
of TSK HW-40S in water
For sugar analysis, PS (0.4 mg) was hydrolysed with 10 M
HC! for 30 min at 80 °C, and the alditol acetates derived
were analysed by GLC-MS using a Hewlett-Packard 5971A
(0.2 mm x 12 m) and temperature program of 8 °min™!
from 150 to 270°C For determination of the absolute
configuration [11,12], LPS (0.8 mg) was subjected to
2-butanolysis [300 uL (R)-2-butanol and 20 uL acetyl
chloride, 100 °C, 3 h]; the products were acetylated and
analysed by GLC-MS as above
Methylation of PS (0.4 mg) was performed by the
Hakomori procedure [13]; products were recovered by
extraction with chloroform/water (1: 1, v/v), hydrolysed
with 10 m HCl for 30 min at 80°C, and the partially
methylated alditol acetates derived were analysed by GLC-
MS as above
NMR spectroscopy
Samples were freeze-dried twice from a 7HO solution and
dissolved in 99.96% “HO "H-NMR and '*C-NMR
spectra were recorded with a Bruker DRX-500 spectrometer
at 60 °C; chemical shifts are reported with internal acetone
(OH 2.225, dc 31.45) as reference Two-dimensional exper-
iments were performed using standard Bruker software
A mixing time of 200 ms was used in TOCSY and HMQC-
TOCSY experiments and 300 ms ina NOESY experiment
Matrix-assisted laser desorption ionization (MALDI) MS
MALDI mass spectra were recorded on a RETOF (time-
of-flight) instrument from Perseptive Biosystems (Framing-
ham, MA, USA) equipped with a pulsed delay source
extractor [14] Spectra were recorded from 256 laser shots
(nitrogen laser, 337 nm) with an accelerating voltage of
20 kV in linear mode For a matrix, 2,5-dihydroxybenzoic
acid was dissolved in aqueous 70% acetonitrile containing
0.1% trifluoroacetic acid Then 1 wL matrix was mixed with
1 uL sample, placed on top of the matrix surface, and
allowed to dry by itself The spectra were calibrated using
insulin (1 pmol-tL~!; m/z 5736) in the same conditions
Mass numbers were rounded to the nearest integer
Rabbit antiserum, antigens and serological techniques
Rabbit antiserum against whole cells of C gillenii O9a,9b
was prepared as described previously [15] LPS of Hafnia
alvei PCM 1186 was from previous studies [15], LPS of
V cholerae Ol was a gift from O Holst (Forschungszen-
trum Borstel, Germany), and that of Escherichia coli O157
A high-molecular-mass PS was isolated by mild acid degradation of LPS of C gillenii 09a,9b followed by gel- permeation chromatography of the carbohydrate portion
on Bio-Gel P-4 Sugar analysis of PS revealed a 4-amino- 4,6-dideoxyhexose as the single monosaccharide constitu- ent This was identified as 4-amino-4,6-dideoxy-p-mannose (p-Rha4N, p-perosamine) by comparison with the corres- ponding authentic samples from LPS of V cholerae O1 [18] using GLC-MS of the alditol acetates and acetylated (R)-2- butyl glycosides
Methylation analysis of PS revealed 4,6-dideoxy-3-O- methyl-4-(N-methyl)acetamidomannose and 4,6-dideoxy- 2-O-methyl-4-(N-methyl)acetamidomannose in the ratio 2:1, which were identified by GLC-MS of partially methylated alditol acetates (retention times 8.98 and 9.03 min, respectively) The former compound was char- acterized by the presence in the mass spectrum of intense ion peaks for the C1-C3, CI-C4, and C4-C6 primary fragments at m/z 190, 275, and 172, respectively The mass spectrum of the latter compound showed intense ion peaks for the fragments Cl-C2, Cl-C4, and C4-C6 at
m/z 118, 275, and 172, respectively Hence, PS is linear
and contains 2-substituted and 3-substituted perosamine residues Further studies showed that PS includes two polysaccharides with the same sugar composition but different structures
The '°C-NMR spectrum of PS (Fig 1, top) contained signals with different integral intensities that could be due to nonstoichiometric O-acetylation (there was a signal for CH;COO at 6 21.5) Some minor signals could belong to the LPS core constituents as they were still present after O-deacetylation of PS with aqueous ammonia The C-NMR spectrum of the O-deacetylated polysaccharide
(PSNH,om Fig 1, bottom) was less complex than the
spectrum of the initial PS and contained signals for several different Rha4NAc residues including signals for anomeric carbons (C1) at 6 101.6—-102.9, carbons bearing nitrogen (C4) at 6 52.9-54.3, CH3-C groups (C6) at 6 18.0-18.3, and N-acetyl groups at 6 23.3—23.5 (CH3) and 175.0-175.7 (CO)
In each carbon group, some signals were two to five times as intense as the single signal
(Table 1) contained, among other things, signals for ano- meric protons (H1) at 6 4.96—5.13, CH3-C groups (H6) at 5 1.17-1.22, and N-acetyl groups at 6 2.04 The two-dimen-
sional COSY and TOCSY spectra of PSNu,on revealed spin
systems for five different Rha4NAc residues, all signals for one of them (Rha4NAc') being about twice as intense as signals for each of four other residues (Rha4NAc"— Rha4NAc’) At the HI co-ordinate, the TOCSY spectrum showed cross-peaks with H2-H6 for Rha4NAc!-Rha4- NAc” but only two cross-peaks, with H2 and H3, for Rha4NAc! At the H6 co-ordinate, the spectrum showed cross-peaks for the whole spin system of each monosaccha- ride residue The COSY spectrum allowed differentiation
Trang 3
SED Ee | —T[TT TT TT TT TT TT TT TTTTT x7 pe ee |
Acetone ©
©
Chemical shift (ppm)
Fig 1 125-MHz '°C-NMR spectra of the initial (PS, top) and O-deacetylated (PSNH,OH:› bottom) polysaccharides from C gillenti O9a,9b Table 1 'H-NMR data Additional chemical shift for the N-acetyl
groups is 6 2.04
Chemical shift (p.p.m.)
Sugar residue HI H2 H35 H4 HS Hồ
O-Deacetylated PS1
> 3)-a-p-Rhap4NAc'-(1 > 4.97 3.85 3.91 3.91 3.84 1.21
~ > 2)-a-p-Rhap4Nac''-(1 > 4.96 3.79 3.99 3.86 3.89 1.20
> 2)-a-p-Rhap4NAc'’-(1 > 5.10 4.13 4.05 3.91 3.80 1.22
~ 3)-a-p-Rhap4NAc’-(1 > 5.03 4.17 3.98 3.99 3.87 1.18
PS2
> 2)-a-p-Rhap4NAcl-(1 > 5.13 4.12 4.03 3.89 3.82 1.17
between protons within each spin system Difficulties
associated with coincidence of signals for some neighbour-
¡ng protons (H3 and H4 of Rha4NAc' and Rha4NAc”)
were overcome using an H-detected 'H,'°C heteronuclear
single-quantum coherence (HSQC) experiment This also
confirmed the assignment for H4 by their correlation to C4
located in the resonance region of carbons bearing nitrogen
(ö 52.9-54.3)
The °C-NMR spectrum of PSNHon (Table 2) was
assigned using a 'H,’°C HSQC experiment The assignment
for C2 was additionally confirmed by a combined 'H,'°C
HMQC-TOCSY experiment (Fig 2), which revealed clear
correlation between H1 and C2 Chemical shifts for C5 (0
69.3-69.6) in the '°C-NMR spectra of PSNu,on and an
a1 — 2-linked p-Rha4NAc homopolymer from JV chole-
rae bio-serogroup Hakata [19] (serogroup O140 [20]) were
close and, hence, all Rha4NAc residues are a-linked (C5 of
B-pyranosides 1s known to resonate 1n a lower field than C5
of o-pyranosides [21]) The relatively low-field position at 6 78.0-79.3 of the signals for C3 of Rha4NAc” and Rha4NAc” and C2 of three other Rha4NAc demonstrated the mode of substitution of the monosaccharides (compare the position at 6 69.0—70.6 of the signals for nonlinked C2 and C3 of Rha4NAc; Table 2)
A NOESY experiment (Fig 3) revealed strong intrares- idue H1/H2 correlations for Rha4NAc’ and Rha4NAc” at
0 5.13/4.12 and 4.97/3.85 and weaker H1/H2 correlations for Rha4NAc ”-Rha4NAc” (the latter are below the level shown in Fig 3) Most importantly, the spectrum contained interresidue cross-peaks between the following transglycos- idic protons: Rha4NAc’ H1/Rha4NAc’ H3 at 5 4.97/3.98,
Rha4NAc" H1/Rha4NAc'* H2 at ồ 5.03/4.13, Rha4NAc'*
HI/Rha4NAc”" H2 at õ 5.10/3.79, and Rha4NAc”” HI/ Rha4NAc” H3 at 6 4.96/3.91 These data are in agreement with the '°C-NMR chemical-shift data and show a Rha4NAc homopolysaccharide with a tetrasaccharide repeating unit (PSINu,on; Fig 4) No interresidue cross- peak was observed for Rha4NAc’ but a strong intraresidue H1/H2 cross-peak at 6 5.13/4.12 and a weak H1/HS cross- peak at 6 5.13/3.82 typical of al — 2-linked sugars with the manno configuration Hence, Rha4NAc’ residues are a1 — 2-linked and build another polysaccharide chain (PS2; Fig 4)
Comparison of the ‘H-NMR, '°C-NMR, and 'H,'°C HMQC spectra of PSNH,on and PS enabled the determi-
nation of the site of attachment of the O-acetyl group In the 'H,'’°C HMOQC spectrum, the intensity of the H2/C2
Trang 4—> 2)-z-D-Rhap4NAc””-(1 > 101.9 79.3 69.0 54.3 69.32 18.0°
PS2
(101.33) (77.86) (68.67) (53.91) (69.33) (17.64)
4° Assignment could be interchanged ° Data from [19] for the O-specific polysaccharide of V cholerae bio-serogroup Hakata (serogroup O140 [20]) are given in parentheses The differences in the chemical shifts are due to the use of different references for calibration (dioxane in the published work [19] and acetone in this work)
ppm
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L675
HI
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5.15 5.10 5.05 5.00 4.95 ppm
Chemical shift (ppm) Fig 2 Part of a 'H,'°C HMQC-TOCSY spectrum of the O-deacetyl-
ated polysaccharide (PSÍNH,on) from C gillenti O9a,9b The corres-
ponding parts of '*C-NMR and 'H-NMR spectra are displayed along
the vertical and horizontal axes, respectively
cross-peak of Rha4NAc” at 5 3.85/70.6 markedly decreased
and a new cross-peak appeared at 0 5.00/72.1 The
'°C-NMR spectrum displayed displacements of parts of
the signals for C1 and C3 of Rha4NAc” from 6 102.9 and
78.0 to 6 101.6 and 76.6, respectively, which are typical of
B-effects of acetylation at O2 [22] Therefore, part of the
Rha4NAc’ residues is O-acetylated at position 2, and PS1
thus has the structure shown in Fig 4 As judged by the
ratio of the integral intensities of the signals for the
O-acetylated and non-O-acetylated residues, the average
degree of O-acetylation of Rha4NAc” in PS1 is + 70% PS2 contains no O-acetyl group
To confirm the existence of two polysaccharides, the carbohydrate portion obtained after mild acid degradation
of C gilleniti O9a,9b LPS was fractionated by gel-perme- ation chromatography on TSK HW-50S to give six fractions (Fig 5) The MALDI mass spectrum of fraction
1 revealed a series of hexose increments with m/z 162, and this fraction was considered to be a glucan-type contami- nant Fraction 4 represented a core oligosaccharide, and fractions 5 and 6 contained low-molecular-mass compounds released from LPS
‘H-NMR and '°C-NMR spectroscopic analysis showed that the perosamine-containing polysaccharides PSI and PS2 were present in fractions 2 and 3, respectively Therefore, the two polysaccharides could be separated and thus belonged to separate LPS molecules
The MALDI mass spectrum of PSI showed a series of ion peaks with differences between ions of 187 or 229 Da, which corresponded to non-O-acetylated and O-acetylated Rha4NaAc, respectively (Fig 6) The low-molecular-mass polysaccharide species (below 4258 Da) were devoid of O-acetyl groups The difference between the ions at m/z
4258 and 4487 corresponded to the O-acetylated Rha4NAc residue (Rha4NAc2Ac’), and the next three peaks in this series at m/z 4674, 4861 and 5048 reflected further chain elongation by non-O-acetylated residues (Rha4NAc”- Rha4NAc’) to complete the tetrasaccharide repeating unit
of PS1 Then, starting from the 1on peak at m/z 5048, the pattern iterated The next ion peaks with a difference of
790 Da for the mono-O-acetylated tetrasaccharide (indicat-
ed by arrows), as well as the intermediate ion peaks (shown
by asterisks), were clearly observed up to m/z 7418 Some of the minor peaks may be due to heterogeneity of the core oligosaccharide The 18 Da difference between 1ons (at 2949 and 2967 m/z and the next peaks in this series) may result from the dehydrated and hydrated forms of 3-deoxy- octulosonic acid (Kdo) residue, respectively, at the reducing end of the polysaccharide
The O-acetylation of PSI begins at a certain polysac- charide chain length (about three tetrasaccharide repeating units) These data are in agreement with the NMR spectroscopic data (see above), which showed that only
~ 70% tetrasaccharide repeating units in PSI are O-acet- ylated
Trang 5Fig 3 Part of a NOESY spectrum of the
Q-deacetylated polysaccharide (PSNH,OH)
from C gillenti O9a,9b The corresponding
parts of the ‘H-NMR spectrum are displayed
along the axes Arabic numerals refer to pro-
tons in sugar residues denoted by roman
numerals as shown in Fig 4
ppm
- 3.70
-3.75
-3.90 1
fg
3.95 9
L 4.05
4.10
4.15 4.20
.20 5.15 5.10 5.05 5.00 4.95 ppm
Chemical shift (ppm)
—>3)-œ-D-Rhap4NAcŸ-(1—›>2)-œ-p-Rhap4NAc'Ÿ-(1—›>2)-œ-D-Rhap4NAc””-(1—›3)-œ-p-Rhap4NAc7-(1—
2
Fig 4 Structures of the polysaccharides (PS1
and PS2) and the O-deacetylated polysacchar-
ide (PS1NH,on) from C gillenti O9a,9b PS2
A
4
RI
) |
6
5
a nan n1 nan T1 Tp TT TT”
Fig 5 Fractionation on TSK HW-S50S of the carbohydrate material
obtained by mild acid hydrolysis of C gillenti O9a,9b LPS For expla-
nation of fractions, see the text
The MALDI mass spectrum of PS2 (not shown)
displayed a series of 10on peaks with a difference between
ions of 187 Da, which corresponded to sequential chain
elongation by one non-O-acetylated Rha4NAc residue The
intensities of the first peaks for the short-chain polysac-
charide species were high and those of the following peaks
decreased, but the series could be traced up to 20 and more
Rha4NAc' residues
R PSinn,on R=H; PS1 R=Ac (~70%) or H
The data obtained suggested that growth of both PSI and PS2 in C gillenii O9a,9b proceeds by sequential transfers of single sugar units A biosynthetic model involving sequential single sugar transfers to the nonreducing end of the growing chain has been suggested for the A-band polysaccharide (b-rhamnan) in Pseudomonas aeruginosa LPS [23] as well as for linear homopolysaccharide O-antigens of Escherichia coli O& and O9 (bD-mannans) and p-galactan I from Klebsiella pneumoniae (reviewed in [24]) This model requires participation of several distinct transferases for the same monosaccharide, as demonstrated for biosynthesis of the A-band polysaccharide [23]
A polysaccharide with the same structure as PS2 has been previously reported to be the O-chain of the LPS of
V cholerae bio-serogroup Hakata [19] (serogroup O140 [20]), whereas PSI is new Interestingly, a polysaccharide
of al — 2-linked and al — 3-linked 4-formamido-4,6-
dideoxy-Db-mannose (N-formyl-Db-perosamine) having a pentasaccharide repeating unit has been found in Brucella melitensis LPS [25] Published structural data [25] do not exclude the occurrence of two separate polysaccharide chains in the LPS of B melitensis The O-chain homo- polymer from Escherichia hermannii LPS composed of
al — 2-linked and ol — 3-linked p-Rhap4NAc residues has been reported to have a pentasaccharide repeating unit containing the tetrasaccharide sequence present in PSI [26]
Trang 6
œ S Y
~ 2 8 %
| | 6 S S š S o Yoo
a ee 1 o RF QA
| Fig 6 Part of a MALDI mass spectrum of
3000 4000 5000 6000 incomplete repeating units are marked by
Mass (m/z) arrows and asterisks, respectively
B
HH
LÔ 4 3 4 1 2 3 4
Fig 7 Silver-stained SDS/PAGE (A) and immunoblotting with anti-
C gillenti O9a,9b serum (B) Lane 1, LPS of Hafnia alvei PCM 1186;
lane 2, LPS of C gillenii O9a,9b; lane 3, O-deacylated LPS of
C gillenii O9a,9b; lane 4, LPS of V cholerae O1
LPS of C gillenii O9a,9b reacted with homologous anti-
O serum in double immunodiffusion (data not shown)
In SDS/PAGE and immunoblotting (Fig 7), anti-(C gille-
nii O9a,9b) serum reacted mainly with slowly moving, high-
molecular-mass LPS species O-Deacylation of C gillenii
O9a,9b LPS had no effect on its serological reactivity From
the separated O-chain polysaccharides, only PS1 reacted in
double immunodiffusion with anti-(C gillenii O9a,9b)
serum, whereas PS2 was inactive, probably, because of a
lower molecular mass
No significant cross-reactivity was observed between anti-
(C gillenii O9a,9b) serum and V cholerae Ol LPS in
double immunodiffusion (not shown) and immunoblotting
(Fig 7) This can be accounted for by different N-acyl substituents at D-Rha4N: N-acetyl or N-[(S)-2,4-dihydroxy- butyryl] group in the O-antigens of C gillenii and V chole- rae [18], respectively The LPS from E coliOQ157, which also contains D-Rha4N [24], also did not react with anti- (C gillenii O9a,9b) serum in double immunodiffusion (data not shown)
ACKNOWLEDGEMENTS
We thank Professor O Holst (Forschungszentrum Borstel, Germany) for the gift of V cholerae Ol LPS and Dr B Maczynska and Professor
A Przondo-Mordarska (Medical Academy, Wroclaw, Poland) for the gift of E coli O157 LPS This work was supported by grant 99-04-
48279 from the Russian Foundation for Basic Research and grant 500- 1-15 from the Polish Academy of Sciences
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