The structure of the O-specific polysaccharide from Ralstonia pickettii

Article abstract of DOI:10.1016/j.carres.2004.05.014

The following structure of the Ralstonia pickettii have been determined using NMR and chemical methods:→4)-α-D-Rha-(1→4)-α-L- GalNAcA-(1→3)-β-D-BacNAc-(1→

Full text of DOI:10.1016/j.carres.2004.05.014

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2045–2047
Note
The structure of the O-specific polysaccharide from Ralstonia pickettii
a,
a
b
b
*
Evgeny Vinogradov, Ludmila Nossova, Anna Swierzko and Maciej Cedzy nꢀ ski
a
Institute for Biological Sciences, National Research Council, 100 Sussex Dr., Ottawa, ON, Canada K1A 0R6
b
The Laboratory of Immunobiology of Infections, Centre for Medical Biology, Polish Academy of Sciences,
Lodowa 106, 93-232 Lodz, Poland
Received 6 May 2004; accepted 19 May 2004
Available online 1 July 2004
Abstract—The following structure of the Ralstonia pickettii have been determined using NMR and chemical methods:
! 4Þ-a-d-Rha-ð1 ! 4Þ-a-l-GalNAcA-ð1 ! 3Þ-b-d-BacNAc-ð1 !
2004 Elsevier Ltd. All rights reserved.
ꢀ
Keywords: Ralstonia; Ralstonia pickettii; LPS; O-Chain; Structure
6
The genus Ralstonia represents aerobic, nonfermentative
Gram-negative bacteria. It was created to accommodate
bacteria from diverse ecological niches previously clas-
from chlorhexidine solutions used for disinfections. It
can degrade numerous toxic compounds.
16
The knowledge of the structure of the O-antigenic
component of lipopolysaccharide (LPS) is helpful in
understanding of molecular details of bacterial biologi-
cal activity. The structure of cell surface polysaccharide
might be determinative factor to the bacterial ability to
adhere to any wet surface for further growing and for-
mation biofilm. This paper includes the results of
structural analysis of the polysaccharide part of the
R. pickettii LPS.
R. pickettii was isolated from purified water-storage
bottle and identified with help of API 20 NE test (Bio-
Merieux, France). Bacteria were cultivated at 37 ꢁC on
normal broth (BTL-Poland). The LPS was extracted by
1
sified as Burkholderia and Alcaligenes with Ralstonia
pickettii (ATCC 27511, formerly called Pseudomonas
pickettii or Burkholderia pickettii) as a type strain.
Recently it was proposed to split this genus into two––
Ralstonia and Wausteria, with R. pickettii remaining in
2
Ralstonia genus. Some members of the genus are
3
important plant pathogens. R. pickettii is the most
common human pathogen of its genus, although other
4
strains were found recently in cystic fibrosis patients.
The virulence of R. pickettii is rather low, however it is
5–10
able to cause bacteremia and nosocomial infections.
Sometimes it infects patients with indwelling intrave-
11
17
nous devices and was isolated from various other
sources.
phenol–water procedure and purified by ultracentrifu-
1;12–14
It is able to grow at a wide range of tem-
gation. The polysaccharide O-chain was isolated from
the LPS by conventional mild acid hydrolysis with
2% HOAc (100 ꢁC, 2 h) followed by Sephadex G50
gel-filtration chromatography of the water soluble
products, yielding a lipid-free high-molecular-mass
peratures (15–42 ꢁC) as well as in the saline solution and
can pass through sterilization filters, thus contaminating
15
sterilized solutions. The bacterium was isolated also
(
O-PS). However no core fraction was observed. GC
Abbreviations: LPS, lipopolysaccharide; GalNA, 2-amino-2-deoxy-
galacturonic acid; BacN, 2,4-diamino-2,4,6-trideoxyglucose (bacillos-
amine).
analysis of alditol acetates prepared from the polysac-
charide showed the presence of rhamnitol and small
amount of glucitol, probably originating from an
impurity.
*
Corresponding author. Tel.: +1-613-990-0832; fax: +1-613-952-9092;
e-mail: evguenii.vinogradov@nrc-cnrc.gc.ca
0
008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.05.014

2046
E. Vinogradov et al. / Carbohydrate Research 339 (2004) 2045–2047
The set of 2D NMR experiments (COSY, TOCSY,
NOESY, HSQC, HMBC) was recorded for the poly-
saccharide sample. The spectra were completely as-
were observed, as well as corresponding HMBC corre-
lations, in agreement with the presented monosacchar-
ide sequence.
18
signed (Fig. 1, Table 1) using Pronto program. This led
to the identification of spin systems of three monosac-
charides, arbitrarily labeled A–C in decreasing order of
their anomeric proton chemical shifts. Monosaccharides
were identified on the basis of vicinal proton coupling
constants and C NMR chemical shifts. Anomeric
configurations were deduced from the J1;2 coupling
constants and chemical shifts of H-1 and C-1 signals.
Position of C-6 signals of uronic acid was found from
HMBC correlations to H-5 protons. Thus the residue A
was identified as 2-acetamido-a-galactopyranosyluronic
acid, residue B as a-rhamnopyranose, and the residue
C as 2,4-diacetamido-2,4,6-trideoxy-b-glucopyranose.
Connections between monosaccharides were identified
on the basis of NOE and HMBC correlations. The NOE
between protons A1 and C3, C1 and B4, B1 and A4
B
A
C
→
4)-α-
D
-Rha-(1→4)-α-
L
-GalNAcA-(1→3)-β-
D
-BacNAc-(1→
1
Minor signals in the NMR spectra of the polysac-
charide were identified as belonging to the terminal
residue of a-GalNAcA (residue A in Table 1). This may
be the result of partial hydrolysis of Rha linkages during
HOAc treatment of the LPS.
Treatment of the polysaccharide with 0.5 M HCl in
MeOH at 70 ꢁC followed by the separation of the
products on reverse phase column gave a number of
products, of which pure oligosaccharide 2, methyl
a-rhamnopyranoside, and a mixture of a-methyl glyco-
sides of GalNAcA (3) and BacNAc (4).
13
0
A
C
ppm
α-GalNAcA6OMe-(1
→
3)-α-BacNAc-(1
→
OMe
B6
C6
2
3
4
5
6
7
0
0
0
0
0
0
2
NMR spectra of compounds 2–4 were completely
assigned (Table 1) and confirmed their structures.
Absolute configuration of Rha was determined by
optical rotation measurement of methyl a-rhamnopyr-
anoside, isolated from the products of the methanolysis
A2
C2 C4
A3 B3 B5
C3
A'4
B2
A5
C5
B4
of polysaccharide. The ½aꢀ value of +52 (c 0.1, water)
A'5
D
A4
was obtained, indicating
for
D
-configuration (lit. ½aꢀ +64.5
80
D
19
D-isomer).
For the determination of the absolute configuration
90
A1
A'1
13
C1
100
10
of two other monosaccharides of repeating unit
NMR chemical shifts were calculated for four possible
combinations of the configurations––DDD
and DLD (assuming that rhamnose is always
data and method described in Ref. 20. The results
C
B1
1
,
DLL, DDL,
5
.0
4.0
3.0
2.0
ppm
D
) using
Figure 1. HSQC spectrum of R. pickettii O-specific polysaccharide.
Table 1. NMR data for the polysaccharide (1) and its methanolysis products (2–4)
Unit, compound
Nucleus
1
2
3
4
5
6
1 fi OMe
6 fi OMe
1
GalNAcA A, 1
H
5.24
98.7
5.24
98.7
5.23
98.4
5.24
98.9
5.16
101.4
4.49
102.5
4.64
99.2
4.76
99.2
4.30
49.9
4.22
49.9
4.16
50.1
4.18
50.0
4.11
70.5
3.95
57.2
4.14
55.5
4.00
55.3
3.99
68.9
3.85
68.1
3.85
67.7
3.86
67.9
3.82
70.0
3.87
77.3
3.92
74.8
3.73
70.1
4.37
75.6
4.29
69.4
4.34
70.4
4.34
70.4
3.44
83.8
3.69
56.5
3.71
56.6
3.65
58.4
4.20
71.8
4.22
71.9
4.46
72.2
4.47
72.2
3.66
68.3
3.66
71.6
3.85
67.7
3.84
68.0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
3
C
173.8
175.7
0
GalNAcA A , 1
H
3
C
GalNAcA A, 2
GalNAcA A, 3
Rha B, 1
H
3.86
54.3
3.86
54.2
3
C
H
3.48
58.5
3
C
H
1.19
17.3
1.24
17.8
1.20
17.9
1.22
3
C
BacNAc C, 1
BacNAc C, 2
BacNAc C, 4
H
3
C
H
3.37
56.5
3.39
56.4
3
C
H
3
C
18.0
0
1
13
A in 1 is nonreducing terminal residue of GalNAcA. Acetate signals in 1: CO 175.9, 174.9, 175.7; Me ( H/ C): 1.99/23.2, 2.02/23.3, 2.05/23.3 ppm.
13
Acetate signals in 2: Me ( H/ C): 1.97/23.3, 1.98/23.2, 2.03/23.4 ppm; in 3: 2.05/23.3 ppm; in 4: 1.97/23.4, 1.98/23.4 ppm.
1

E. Vinogradov et al. / Carbohydrate Research 339 (2004) 2045–2047
2047
showed best fit between calculated and experimental
data for -BacN and -GalNA. This conclusion agrees
with the observed optical rotation of the polysaccharide
MeOH (2.0 mL), cooled in a dry ice/acetone bath, fol-
lowed by addition of acetyl chloride (0.1 mL). The dis-
solved material was kept at 70 ꢁC for 24 h, dried in an air
stream, and the products were separated by HPLC with
UV detection (220 nm) on a C18 column (Phenomenex
Aqua, 10 · 250 mm) using a gradient of water to 90%
MeCN. Fractions were also analyzed for the presence of
sugars by charring spots on TLC plate after dipping in
D
L
½
aꢀ )53 (c 1, water), which is to be expected qualita-
D
tively for the sum of two negative values contributed by
a-
Rha. The optical rotation of the disaccharide 2 was )16
c 0.2, water) due to canceling of positive contribution of
a- -BacN and negative of a- -GalNA; it would have
L
-GalNA and b-
D
-BacN, and one positive from a-
D-
(
D
L
2 4
5% H SO in MeOH.
large absolute value in case of identical configuration of
both monosaccharide residues.
The structure of the R. pickettii polysaccharide
includes a fragment:
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2
4
(ꢁ5 mg, 1 h), neutralized with
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1
.2. Preparation of the polysaccharide fragments
2
For the preparation of methyl a-rhamnopyranoside and
compounds 2–4, a sample (40 mg) was suspended in dry