Structural and serological studies on the O-antigen show that Citrobacter youngae PCM 1505 must be classified to a new Citrobacter O-serogroup

Article abstract of DOI:10.1016/j.carres.2012.06.010

The O-polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter youngae PCM 1505 was studied by sugar and methylation analyses along with 1D and 2D ¹H and ¹³C NMR spectroscopies. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: Structural and serological data obtained earlier and in this work show that the strain studied is a candidate to a new Citrobacter O-serogroup.

Full text of DOI:10.1016/j.carres.2012.06.010

Carbohydrate Research 360 (2012) 52–55
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Note
Structural and serological studies on the O-antigen show that Citrobacter
youngae PCM 1505 must be classified to a new Citrobacter O-serogroup
Ewa Katzenellenbogen , Nina A. Kocharova , Sabina Górska-Fr a˛ czek , Andrzej Gamian a,c,
a,
⇑
b
a
b
b
Alexander S. Shashkov , Yuriy A. Knirel
a
L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wrocław, Poland
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow 119991, Russian Federation
Department of Medical Biochemistry, Wrocław Medical University, 50-368 Wrocław, Poland
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 May 2012
Received in revised form 12 June 2012
Accepted 16 June 2012
Available online 22 June 2012
The O-polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter youngae
PCM 1505 was studied by sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spec-
troscopies. The following structure of the tetrasaccharide repeating unit of the polysaccharide was estab-
lished:
Keywords:
Citrobacter youngae
Lipopolysaccharide
O-Polysaccharide structure
O-Antigen
Serological classification
Structural and serological data obtained earlier and in this work show that the strain studied is a can-
didate to a new Citrobacter O-serogroup.
Ó 2012 Elsevier Ltd. All rights reserved.
Gram-negative bacteria of the genus Citrobacter from the family
Enterobacteriaceae are opportunistic pathogens often responsible
for serious enteric diseases as well as extraintestinal disorders,
Now, we report on a new structure of the OPS of Citrobacter
youngae PCM 1505. This strain was originally classified to sero-
group O6 as IHE Be 16/50 = M.W. Wright strain ‘Mich 11’. Later,
1
such as neonatal meningitis and brain abscesses. Currently, the
the pathogenic strain ‘Mich 11’ initially isolated from stools was
genus comprises 11 named genomospecies,1 and known Citrobac-
reclassified as a strain of C. youngae serogroup O8.
12,13
The data
2
ter strains are classified into 43 O-serogroups and 20 chemotypes
on the OPS structure combined with serological data showed that,
in fact, strain PCM 1505 belongs to neither of the serogroups.
The LPS was isolated from dry bacterial cells by extraction with
hot aqueous phenol. Mild acid degradation of the lipopolysaccha-
ride afforded an OPS, which was isolated by GPC on Sephadex
G-50. Sugar analysis by GLC of the acetylated alditols derived after
based on their serological cross-reactivity and chemical composi-
tion of the lipopolysaccharide (LPS) O-antigens.3 So far the
structures of more than 30 different Citrobacter O-antigens
,4–11
(
O-polysaccharides, OPSs) have been elucidated.1
Chemical
and serological studies on Citrobacter LPSs are carried out with
the aim to understand the molecular basis for cross-reactivity be-
tween representatives of various enterobacterial genera and to im-
prove the serological classification of this highly heterogenous
group of bacteria.
3 2
hydrolysis of the OPS with 2 M CF CO H revealed galactose and
rhamnose in the ratio ꢀ1:2.9. GLC of the acetylated (S)-2-octyl gly-
cosides showed that rhamnose has the
with -galactose oxidase confirmed the
D
configuration. Oxidation
D
D configuration of galact-
ose. The content of galactose determined colorimetrically in the
OPS hydrolysate was 27.5%. Methylation analysis demonstrated
the presence of 2-substituted, 3-substituted, 3,4-disubstituted
⇑
Corresponding author. Tel.: +48 71 3371172; fax: +48 71 3709975.
E-mail address: katzenel@iitd.pan.wroc.pl (E. Katzenellenbogen).
0
008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.06.010

E. Katzenellenbogen et al. / Carbohydrate Research 360 (2012) 52–55
53
rhamnose and terminal galactofuranose in the ratio 0.98:0.87:
.0:0.81 or 0.7:1.0:1.0:0.75 when 2 M CF CO H or 10 M HCl were
Gal C-1, RhaII H-4 at d 5.08/81.3; 4.80/78.2; 102.5/4.13; 97.9/
4.03, and 103.2/3.66, respectively. These data defined the sequence
of the monosaccharides in the repeating unit, and hence the OPS of
C. youngae PCM 1505 has the structure shown in Chart 1.
1
3
2
used for hydrolysis, respectively. Therefore, the OPS has a branched
tetrasaccharide repeating unit with galactofuranose as a lateral
monosaccharide residue.
The OPS studied shares the rhamnose backbone structure
with that of C. braakii (formerly C. freundii) PCM 1536 from
The H NMR and 13C NMR (Fig. 1) spectra of the OPS showed,
1
1
6
inter alia, signals for four anomeric atoms at d
7.9–103.7, three major CH -C groups of rhamnose residues
1.33–1.35 and d 18.1–18.7, and one HOCH –C
group of galactose at d 63.6. The four major sugar spin systems
were assigned by tracing connectivities in the 2D H, H COSY, TOC-
H
4.80–5.29 and d
C
serogroup O8. The two OPSs differ only in the lateral sugar
residue (Chart 1), which is -galactofuranose in strain PCM 1505
or -xylofuranose in strain PCM 1536.
Immunoblotting was carried out to check if there is any serolog-
9
3
D
I
III
(
Rha –Rha ) at d
H
C
2
D
C
1
1
ical relationship between C. youngae PCM 1505 and C. braakii PCM
1536 (serogroup O8). No cross-reaction was observed between the
LPSs of the two strains and either anti-C. youngae PCM 1505 or
anti-C. braakii PCM 1536 sera (Fig. 2). Earlier, it has been demon-
strated that C. youngae PCM 1505 and PCM 1504 must not be
classified to serogroup O6 together with C. braakii PCM 1531.6
Therefore, the chemical and serological data show that C. youngae
PCM 1505 does not belong to any existing Citrobacter O-serogroups
and must be reclassified to a new serogroup.
1
13
SY, and H, C HSQC spectra (Table 1).
3
J
H,H coupling constants estimated from the 2D NMR spectra
demonstrated that all rhamnose residues are in the pyranose form
and confirmed that galactose is in the furanose form. The C-5
II
III
chemical shifts of d 69.1 and 70.7 indicated that Rha and Rha
I
are
7
a
-linked, whereas Rha characterized by the value for C-5 of d
14
4.2 is b-linked (compare published data d 70.0 for
a-Rhap and
I
d 73.7 for b-Rhap). The b configuration of Rha was confirmed by
intraresidue correlations of H-1 with H-3 and H-5 in the 2D ROESY
spectrum of the OPS. The C-1 chemical shift of d 103.2 showed that
1
1
. Experimental
Gal is
reported for methyl
Low-field positions of the signals for C-2 of Rha , C-3 of Rha ,
and C-3 and C-4 of Rha at d 78.1–81.3, as compared with their
a-linked (the values for C-1 of d 103.8 and 109.9 have been
1
5
a
- and b-galactofuranosides, respectively).
.1. Bacterial strain, cultivation, isolation of the
I
III
lipopolysaccharide and O-polysaccharide
II
positions at d 71.3–73.5 in the spectra of the corresponding ano-
C. youngae PCM 1505 and C. braakii PCM 1536 were from the
collection of the L. Hirszfeld Institute of Immunology and Experi-
mental Therapy (Wrocław, Poland). Strain PCM 1505 was classified
originally as a member of serogroup O6 (IHE Be 16/50 = M.W.
Wright strain ‘Mich 11’ = PCM 1505 O6:16) but later was reclassi-
mers of non-substituted rhamnopyranose,14 revealed the glycosyl-
II
ation pattern in the repeating unit and showed that Rha is located
at the branching point. In accordance with the terminal position of
Gal, the C-2 to C-6 chemical shifts of this residue were close to
those in the non-substituted monosaccharide.15
In the 2D ROESY spectrum of the OPS, there were a number of
interresidue correlations, from which the correlations between
the transglycosidic protons were assigned as follows, taking into
account the positions of substitution of the monosaccharide resi-
1
3
fied to serogroup O8 as C. youngae O8:32,33, and is present in the
1
2
Lanyi collection as serotype O8a,1c;32,33. Strain PCM 1536 (IHE
Be 64/57) was classified as C. braakii O8a,8b:35,37. Bacteria were
1
7
cultivated in a liquid medium as described, then harvested and
freeze-dried.
II
I
III
II
I
III
dues: Rha H-1, Rha H-2; Rha H-1, Rha H-3; Rha H-1, Rha
LPS of strain PCM 1505 was isolated from bacterial cells by the
II
1
8
H-3, and Gal H-1, Rha H-4 at d 5.07/4.13; 5.08/4.15; 4.80/4.03,
and 5.29/3.66, respectively. Accordingly, the H, C HMBC spec-
trum showed the following correlations between the anomeric
protons and linkage carbons and vice versa: Rha H-1, Rha C-3;
Rha H-1, Rha C-3; Rha C-1, Rha H-2; Rha C-1, Rha H-3, and
phenol–water procedure, recovered from the water phase, and
1
13
1
9
purified as described. The yield of the LPS was 1.7% of dry bacte-
rial mass. A sample of the LPS (270 mg) was heated with 1% HOAc
III
II
(
27 mL) for 1.5 h at 100 °C, and a lipid precipitate was removed by
I
III
II
I
I
III
centrifugation. The carbohydrate-containing supernatant (61% of
Figure 1. 13C NMR spectrum of the OPS from C. youngae PCM 1505. RI-RIII indicate Rha –RhaIII, G indicates Gal.
I

5
4
E. Katzenellenbogen et al. / Carbohydrate Research 360 (2012) 52–55
Table 1
1
H and 13C NMR chemical shifts (d, ppm) of the O-polysaccharide of C. youngae PCM 1505
Sugar residue
Nucleus
1
2
3
4
5
6 (6a, 6b)
I
1
?
?
?
2)-b-
3,4)-
3)-
-Galf-(1?
D
-Rhap -(1?
H
C
4.80
97.9
5.07
102.5
5.08
103.7
5.29
103.2
4.13
78.1
4.19
71.5
4.17
68.6
4.05
77.9
3.73
74.7
4.15
81.3
4.03
78.2
4.07
76.0
3.45
73.6
3.66
79.5
3.55
72.0
3.71
82.0
3.42
74.2
4.34
69.1
3.90
70.7
3.79
74.7
1.33
18.1
1.35
18.7
1.33
18.4
3.60, 3.73
63.6
1
1
1
1
1
1
1
3
II
a
-
D
-Rhap -(1?
H
3
C
a
-
D
-RhapIII-(1?
H
3
C
a-
D
H
3
C
À1
III
II
I
to 270 °C at 8 °C min . The absolute configuration of rhamnose
was determined by GLC of the acetylated (S)-2-octyl glycosides20
under the same conditions. The absolute configuration of galactose
3
)- -D-Rhap -(1 3)- -D-Rhap -(1 2)- -D-Rhap -(1
4
2
1
was established using
D-galactose oxidase after hydrolysis of the
1
OPS with 2 M CF CO H (120 °C, 2 h).
Methylation of the OPS was performed as described. The par-
tially methylated monosaccharides were derived by hydrolysis of
the methylated polysaccharide with 2 M CF
0 M HCl (80 °C, 30 min), converted into the alditol acetates, and
3
2
-
D-Galf
2
2
3
)- -D-Rhap-(1 3)- -D-Rhap-(1 2)- -D-Rhap-(1
4
3 2
CO H (120 °C, 2 h) or
1
1
analyzed by GLC–MS on a Hewlett-Packard 5971A instrument as
in sugar analysis.
-
D-Xylf
Chart 1. Structures of the OPSs from C. youngae PCM 1505 (this work) and C. braakii
PCM 1536 (serogroup O8).16
1.3. NMR spectroscopy
An OPS sample was freeze-dried twice from a 99.9 % D
2
O solu-
O. H and C NMR spectra were re-
corded at 53 °C on a Bruker DRX-500 spectrometer (Germany)
using internal acetone (d 2.225, d 31.45) as reference. 2D NMR
1
13
tion and dissolved in 99.95% D
2
H
C
spectra were obtained using standard Bruker software, and Bruker
XWINNMR 2.6 program was employed to acquire and process the
NMR data. A mixing time of 200 ms was used in ROESY and TOCSY
experiments.
1
.4. SDS–PAGE and immunoblotting
SDS–PAGE of the LPSs was performed by the method of Lae-
2
3
24
mmli. The gels were stained using the silver reagent. Rabbit
antisera against Citrobacter strains PCM 1505 and PCM 1536 (sero-
1
7
group O8) were prepared as described. Immunoblotting was car-
2
5
ried out as previously. Briefly, after separation by SDS–PAGE, the
LPSs were transblotted from the gel onto an Immobilon P (Milli-
pore Corp., Bedford, MA, USA) membrane. The transblot was incu-
bated with antiserum, washed with Tris-buffered saline (20 mM
Tris/HCl, 50 mM NaCl, pH 7.5), and incubated with alkaline phos-
phatase-conjugated goat anti-(rabbit IgG). The immunoblot was
visualized with a staining reagent (nitro-blue tetrazolium and 5-
bromo-4-chloro-3-indolyl phosphate in 0.05 M Tris/HCl, pH 9.5,
Figure 2. Silver-stained SDS–PAGE (A) and immunoblotting of the LPSs of C. braakii
PCM 1536 (lane 1) and C. youngae PCM 1505 (lane 2) with anti-C. youngae PCM 1505
serum (B) and anti-C. braakii PCM 1536 (serogroup O8) serum (C).
2
containing 5 mM MgCl ).
Acknowledgements
the LPS mass) was fractionated by GPC on a column (100 Â 2.0 cm)
of Sephadex G-50 Fine (Sigma) in 0.05 M pyridine/HOAc buffer pH
This work was supported by the Federal Targeted Program for
Research and Development in Priority Areas of Russia’s Science
and Technology Complex for 2007–2013 (State Contract No.
16.552.11.7050) using the equipment of the Center for Collective
Use EBEE of the FGBOU VPO MarGTU.
5
.6, and monitored using the phenol–sulfuric acid reaction. A high-
molecular mass OPS was obtained in a yield of 43% of the total
amount of carbohydrates eluted from the column.
1
.2. Sugar and methylation analyses
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