Tài liệu Báo cáo Y học: Structure of the O-polysaccharide and classification of Proteus mirabilis strain G1 in Proteus serogroup O3 potx

Structure of the O-polysaccharide and classification
of
Proteus mirabilis
strain G1 in
Proteus
serogroup O3
Zygmunt Sidorczyk
1
, Krystyna Zych
1
, Filip V. Toukach
2
, Nikolay P. Arbatsky
2
, Agnieszka Zablotni
1
,
Alexander S. Shashkov
2
and Yuriy A. Knirel
2
1
Department of General Microbiology, Institute of Microbiology and Immunology, University of Lodz, Poland;
2
N.D. Zelinsky
Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
The O -chain polysaccharide of the lipopolysaccharide (LPS)
of a previously nonclassi fied strain o f Proteus m irabilis
termed G1 was studied by sugar analysis and
1
Hand

13
C
NMR spectroscopy, including 2D COSY, TOCSY, rota-
ting-frame NOE (ROESY), H-detected
1
H,
13
CHMQC,and
heteronuclear multiple-bond correlation (HMBC) experi-
ments. The following structure of the polysaccharide was
established:
where
D
-GalA6(
L
-Lys) stands for N
a
-(
D
-galacturonoyl)-
L
-lysine. The structure of the O-polysaccharide of
P. mirabilis G1 is similar, but not identical, to that of
P. mirabilis S1959 and OXK belonging t o serogroup O3.
Immunochemical studies with P. mirabilis G1 and S1959
anti-(O-polysaccharide) sera revealed close L PS-based
serological relatedness of P. mirabilis G1 and S1959, and
therefore it was suggested to classify P. mirabilis G1 in
serogroup O3 as a subgroup. P. mirabilis G1 and S1959
anti-(O-polysaccharide) sera also cross-reacted with LPS

of P. mirabilis strains from two other serogroups contain-
ing
D
-GalA6(
L
-Lys) in the O-polysacch aride or in the core
region.
Keywords: Proteus mirabilis; O-polysaccharide; lipopoly-
saccharide; N
a
-(
D
-galacturonoyl)-
L
-lysine; serogroup.
Much has been written about the taxonomy o f Proteus since
the original pub lication by Hauser in 1885 who established
the genus [1]. Currently, the genus Proteus consists of
five named species (P. mirabilis, P. penneri, P. vulgaris,
P. myxofaciens and P. hauseri) and three unnamed
genomospecies 4, 5 and 6 [2,3]. Proteus rods are widespread
in the environment a nd make up part of the normal flora of
the human gastrointestinal tract. Proteus ran ks third (after
Escherich ia and Klebsiella) as the cause of uncomplicated
cystitis, pyelonephritis and prostatitis, particularly, in hos-
pital-acquired cases [4]. P. mirabilis accounts for approxi-
mately 3% of nosocomial infections in the United States
where, together w ith P. penneri, it may play a role in s ome
diarrhoeal diseases [5]. Recently, it has been suggested that
P. mirabilis may p lay an e thiopathogenic role in rheumatoid

arthritis [6].
According to the serological specificity of the O-chain
polysaccharides (O-antigens) of the lipopolysaccharides
(LPS), strains of P. mirabilis and P. vulgaris have been
classified into 6 0 O-serogroups [7,8], i ncluding 49 numbered
serogroups (O1 to O49) [7]. Recently, immunochemical
studies of LPS enabled establishment of a number of
additional serogroups for P. penneri strains [9–11]. The
serological heterogeneity of Proteus strains is associated
with a high diversity of the O-antigen composition and
structure [12,13]. A common structural f eature of most
Proteus O-antigens studied so far is the presence of
hexuronic acids and their amides with amino acids, which
often serve as immunodominant g roups [13].
Here, we report on the structure of a new acidic
O-polysaccharide from a nonclassified strain P. mirabilis
termed G1, which contains an amide of
D
-galacturonic acid
with
L
-lysine. Based on chemical an d serological data, we
propose to classify th is strain in Proteus serogroup O3.
MATERIALS AND METHODS
Bacterial strains and growth
P. mirabilis strains G1 and D52 were kindly provided by
J. Gmeiner (Institute for Microbiology and Genetics,
Darmstadt, Germany). Strain G1 was a clinical isolate
from urine of a woman with bacteriuria and could be
classified in none of 49 O-serogroups in the Kaufman–

Perch scheme of Proteus [7]. Biochemical properties of both
strains were checked in API 20E test, which showed 99.9%
identity with the P. mirabilis species. For other s trains used
in this work, P. mirabilis O28 (51/57) was purchased from
the Czech National Collection of Type Cultures ( CNCTC,
Institute of Epidemiology and Microbiology, Prague,
Czech Republic), and P. mirabilis S1959 (O3) and its R14
mutant (T-like form) came from the collection of the
Correspondence to Z. Sidorczyk, D epartment of Ge neral Microbio-
logy, Institute of Microbiology and Immun o logy, University of Lodz,
90–237, Lodz, Poland. Fax a nd Tel.: + 48 42 635 44 67,
E-mail:
Abbreviations: EIA, enzyme i mmunosorbent assay;
D
-GalA(l-Lys),
N
a
-(
D
-galacturonoyl)-
L
-lysine;
D
-GlcA,
D
-glucuronic acid; HMBC,
heteronuclear multiple-bond correlation; LPS, lipopolysaccharide;
ROESY, rotating-frame NOE spectroscopy.
(Received 15 O ctober 2001, revised 2 January 2002, accepted
11 January 2002)

Eur. J. Biochem. 269, 1406–1412 (2002) Ó FEBS 2002
Institute of Microbiology and Immunology, University of
Lodz, Poland.
Dry bacteria w ere obtained from aerated liquid cultures
as described [14].
Isolation and degradation of lipopolysaccharide
LPS were obtained by extraction of b acterial mass with a
hot phenol/water mixture [15] and purified by treatment
with aqueo us 5 0% CC l
3
CO
2
Hat4°C followed by dialysis
of the supernatant. Alkali-treated LPS were prepared by
saponification of LPS with 0.25
M
NaOH ( 56 °C, 2 h)
followed by precipitation with ethanol.
Acid degradation of P. mirabilis G1 LPS was performed
with 0.1
M
NaOAc/HOAc buffer pH 4.5 at 100 °Cfor
1.5 h. The O-polysaccharide was isolated by gel-permeation
chromatography on a column (3 · 65 cm) of Sephadex
G-50 (Pharmacia) using 0.05
M
pyridinum-acetate buffer
pH 4.5 as eluent; monitoring was p erformed using a Knauer
differential refractometer (Germany).
Anti-(O-polysaccharide) sera

Rabbit polyclonal anti-(O-polysaccharide) sera against
P. mirabilis G1 and P. mirabilis S1959 were obtained by
intravenous imm unization of rabbits every 5 days with 0.25,
0.5 and 1.0 mL bacterial suspension ( 1.5 · 10
10
c.f.u.ÆmL
)1
),
boiled at 100 °C f or 2 h. One week after t he last injection,
rabbits were bled. The obtained antisera were stored at
)20 °C. For passive immunohemolysis, the antisera w ere
inactivated a t 56 °C f or 30 min and absorbed with sheep red
blood cells. O ne hemolytic unit of anti-(O-polysaccharide)
serum was defined as the antibody dilution yielding 50%
lysis of sheep red b lood cells.
Agglutination test
Agglutination i n tubes was performed w ith a suspension of
heat-killed Proteus bacteria incubated (24 h at 50 °C) with
diluted P. mirabilis G1 anti-(O-polysaccharide) serum.
Passive immunohemolysis, inhibition of passive
immunohemolysis and absorption
Sheep red blood cells were sensitized with a growing
concentration of a lkali-treated LPS fo r 30 min at 37 °C, then
washed with NaCl/P
i
pH 7.2 (15 m
M
Na
2
HPO

4
,150m
M
NaCl) and suspended at a concentration 0 .5% in veronal
buffer pH 7.3 (1.8 m
M
sodium 5,5-diethylbarbiturate,
3.1 m
M
5,5-diethylbarbituric acid, 150 m
M
NaCl, 0.5 m
M
MgCl
2
,0.15 m
M
CaCl
2
). Anti-(O-polysaccharide) serumwas
serially twofold diluted with 50 lL veronal buffered saline
and, after adding 50 lL antigen suspension and 25 lL
guinea-pig complement diluted ( 1 : 20) with veronal buffer,
the plate was incubated a t 3 7 °C f or 1 h. T he last dilution of
antiserum g iving 50% hemolysis was established as a titre.
A total of 25 lL anti-(O-polysaccharide) serum c ontain-
ing 2 or 3 h emolytic units of antibodies was incubated with
25 lL twofold serially diluted inhibitor in microtitrate
plates. After incubation (15 min, 37 °C), 50 lLsensitized
sheep red blood cells and 25 lL complement were added,

the plate was incubated (37 °C for 1 h) and the 50%
inhibition value of hemolysis was read.
In the absorption test, 1 mL anti-(O-polysaccharide)
serum d iluted with N aCl/P
i
(1 : 50) was treated with 100 lL
sheep red blood cells (0.2 mL) sensitized w ith the respective
antigen (200 lgLPS)for30mininanicebath.After
centrifugation, the level of antibodies was evaluated using
passive immunohemolysis test.
Enzyme immunosorbent assay (EIA) and inhibition
of the reaction in EIA
Maxi Sorb microtiter plates (U-bottom form, Nunc,
Denmark) were coated with LPS (50 ng per well) diluted
with NaCl/P
i
at 4 °C for 16 h and washed with water. Plates
were blocked with 2.5% casein in NaCl/P
i
(incubation with
NaCl/P
i
/casein for 1 h at 37 °C followed by two washing
cycles with NaCl/P
i
) and anti-(O-polysaccharide) serum
diluted appropriately with NaCl/P
i
/casein was added. After
incubation at 37 °C for 1 h and washing, peroxidase-

conjugated goat anti-(rabbit IgG) Ig (Sigma) diluted
1 : 1000 with NaCl/P
i
/casein was added and incubation
was continued for 1 h at 37 °C. After washing in NaCl/P
i
,
the plates were washed twice in substrate buffer (0.1
M
sodium citrate pH 4 .5). The substrate solution was freshly
prepared as follows: 1 mg azino-di-3-ethyl-benzthiazolin-
sulfonic acid (Sigma) was dissolved in 1 mL of substrate
buffer with ultrasonication for 3 min and then 25 lL0.1%
H
2
O
2
was added. After incubation for 30 min at 37 °C, the
reaction was stopped by adding aqueous 2% oxalic acid,
and t he plates were read using an Easy Beam Reader (SLT
Lab instruments, Finland) at 405 nm. The end titre was
taken as the highest dilution of antiserum yielding
A
405
>0.2.
Inhibitor w as serially twofold diluted with 30 lLNaCl/
P
i
/casein and mixed in V-shaped microtitrate plates (Med-
lab, Poland) with an equal volume of antibodies diluted

with the same buffer to give A
405
of 1.0–1.6 without adding
the inhibitor. After incubation at 37 °C for 15 min, the
mixture was transf erred t o EIA plates coated with LPS, and
further steps were performed a s described above.
SDS/PAGE and Western blot
SDS/PAGE and W estern immunoblots w ere carried out
according to Laemmli [16]. Briefly, LPS in sample buffer
(4 lL per lane) were separated using 3.5% polyacrylamide
stacking gel and 12.5% running gel a nd then transferred to
a nitrocellulose membrane. The membrane was blocked
with 10% s kimmed milk in dot-blot buffer pH 7.4 (50 m
M
Tris/HCl and 200 m
M
NaCl)at20 °C for 1 h and incubated
with anti-(O-polysaccharide) serum diluted 1 : 300 with the
same buffer for 16 h. The reaction was developed with
alkaline phosphatase-conjugated goat anti-(rabbit IgG) Ig
(Dianova, Germany) diluted 1 : 500 with blotting buffer
supplemented with dried skim milk at 20 °Cfor2h.
5-Bromo-4-chloro-3-indoylphosphate p-toluidine and
p-nitroblue tetrazolium chloride (Bio-Rad, Poland) were
used as substrate.
Sugar analysis
The polysaccharide was hydrolysed with 3
M
CF
3

CO
2
H
(100 °C, 4 h), amino and neutral sugars were identified
using Biotronik L C-2000 amino-acid a nd s ugar analysers a s
Ó FEBS 2002 O-polysaccharide of Proteus mirabilis G1 (Eur. J. Biochem. 269) 1407
described [17]. The absolute configurations of the mono-
saccharides were determined by GLC of the acetylated
(S)-2-butyl glycosides [18,19] using a Hewlett-Packard 5890
chromatograph equipped with an Ultra 2 capillary column
and a temperature gradient of 160–290 °Cat3°CÆmin
)1
.
NMR spectroscopy
Samples were deuterium-exchanged by freeze-drying two
times from D
2
O and examined in D
2
Oat45°Cusing
internal acetone as reference (d
H
2.225, d
c
31.45).
1
Hand
13
C
NMR spectra were recorded with a Bruker DRX-500

spectrometer equipped with an SGI INDY computer
workstation. 2D NMR experiments were performed using
standard Bruker software, and
XWINNMR
program (Bruker)
was used to acquire and process data. A mixing time of 200
and 300 ms was used in TOCSY and ROESY experiments,
respectiv ely.
RESULTS AND DISCUSSION
Structural studies
The O-polysaccharide was prepared by mild acid degrada-
tion of P. mirabilis G1 LPS followed by gel-permeation
chromatography on Sephadex G-50. Sugar analysis of the
polysaccharide after a cid hydrolysis revealed glucuronic a cid
(GlcA)andgalacturonicacid(GalA)intheratio 1:5.
Analysis on an amino-acid analyser showed the presence o f
2-amino-2-deoxygalactose and lysine. The
D
configuration
of GalA and GalN was determined by GLC of the
acetylated (S)-2-butyl glycosides and the
L
configuration of
lysine by GLC of the acetylated (S)-2-butyl ester. The
D
configuration of GlcA was established b y a nalysis of
13
C
NMR chemical shift data of the polysaccharide (see below).
The

13
C NMR spectrum of the polysacc haride (Fig. 1 )
contained signals for four anomeric carbons at d 100.9–
105.5, one nonsubstituted (d 62.4) and one substituted (d
66.7) C-CH
2
OH groups (C6 of GalN, data of attached-
proton test [20]), two carboxyl groups at d 172.3 a nd 174.6
(C6 of GlcA and GalA), two carbons bearing n itrogen at d
52.3 and 53.2 ( C2 of GalN), 14 sugar ring carbons bearing
oxygen in t he region d 69.0–81.3, two N-acetyl groups (CH
3
at d 23.6, C O a t d 175.9 and 176.2), and six carbons of lysine
at d 23.0, 27.4, 31.9, 40.5, 54.3 a nd 177.9 (Table 1, compare
published data [21–23]). Accordingly, the
1
HNMRspec-
trum of the polysaccharide (Fig. 2) contained s ignals for
four anomeric protons at d 4.51–5.20, two N-acetyl groups
at d 2.02 and 2.05, and signals for lysine as shown in
Table 1. Therefore, t he polysaccharide has a tetrasaccharide
repeating unit containing one residue each of
D
-GlcA and
D
-GalA, two residues o f
D
-GalNAc, and
L
-lysine. A smaller

than expec ted relative con tent o f GlcA i n t he polysaccharide
hydrolysate could be accounted for by its retention in
oligosaccharides with GalN (see the polysaccharide struc-
ture below).
The
1
Hand
13
C NMR spec tra o f the polysaccharide w ere
assigned using 2D COSY, TOCSY, ROESY,
1
H,
13
C
HMQC, and HMQC-TOCSY experiments (Table 1 ). The
TOCSY spectrum showed correlations between H1 and
H2–H5 for GlcA and GalA and between H1 and H2–H4
for both GalNAc residues (GalNAc
I
and GalNAc
II
). The
signals for H5 and H6 of GalNAc
I
were assigned by H4/H5
correlation in the R OESY spectrum a nd H5/H6 c orrelation
in the COSY spectrum. The corresponding
13
CNMR
signals were found by

1
H,
13
C correlations in the HMQC
spectrum, and three remaining signals were assigned to
H5/C5, H6a/C6, and H6b/C6 correlations of GalNAc
II
.
J
H,H
coupling constant values estimated from the 2D
COSY an d T OCSY spectra were typical of sugars with the
gluco and galacto configurations in the pyranose form [24].
The GlcA residue was identified by a large J
3,4
coupling
constant value of  10 Hz, as compared with J
3,4
£ 3Hz
for t he other sugars that have the ga la cto configuration. The
Fig. 1. 125-MHz
13
C NMR spectrum of the O-polysaccharide of P. mirabilis G1.
1408 Z. Sidorczyk et al. (Eur. J. Biochem. 269) Ó FEBS 2002
GalNAc residues were distinguished by correlation
of protons at carbons bearing nitrogen (H2) to the
corresponding carbons (C2) revealed by the
1
H,
13

CHMQC
experiment. The signals for the carboxyl groups (C6 of
GlcA and GalA and C1 of Lys) were assigned by H5/C6
and H2/C1 correlations, respectively, observed in the
HMBC spectrum. The spectrum showed also a correlation
between H2 o f Lys and C 6 o f G alA, thus demonstrating th e
presence of N
a
-galacturonoyllysine (GalA6Lys). This con-
clusion was confirmed by t ypical
13
C NMR chemical shifts
for the free carboxyl group of lysine (d 177.9) and the
amidated carboxyl group of GalA (d 172.3) (compare
published data [21,23]).
Relatively large J
1,2
coupling constant values of 7–8 Hz
determined from the
1
H NMR spectrum for the anomeric
protons at d 4.51–4.55 showed that GlcA and both GalNAc
residues are b-linked. The a-linkage was suggested for a
poorly resolved H1 signal of GalA that appeared downfield
at d 5.20, and was confirmed by t he
13
C NMR chemical shift
data (Table 1, compare to published data [23]).
Significant d ownfield displacements of the signals for C3
of GalNAc

I
,C4ofGlcA,C4andC6ofGalNAc
II
to d 81.3,
81.3, 75.9, and 66.7, respectively, as compared with their
positions in the corresponding nonsubstituted sugars [25],
demonstrated the glycosylation pattern. The
13
CNMR
chemical shifts for the GalA residue were close t o t hose for
the nonsubstituted monosaccharide [23] and, hence, this
residue is terminal.
The ROESY s pectrum showed a GalA H1/GalNAc
II
H4
correlation at d 5.20/4.03, and, hence, GalALys is attached
to the disubstituted GalNAc
II
residue as a m onosaccharide
side chain. Correlations of the b-linked sugars in the main
chain were d ifficult to i nterpret because o f close positions of
the H1 r esonances and m ultiple c oincidences of i ntraresidue
H1/H3,H5 and interresidue cross-peaks. The HMBC spec-
trum contained a GalNAc
I
H1/GalNAc
II
C6 at d 4.55/66.7
and two overlapping cross-peaks at 4.51–4.53/81.3, which
could be assigned to GalNAc

II
H1/GlcA C4 and GlcA H1/
GalNAc
I
C3 correlations. In addition, GalA C1/GalNAc
II
H4 and GalNAc
II
C1/GlcA H4 cross-peaks were present at
Table 1. 500-MHz
1
H and 125-MHz
13
C NMR ch emical shifts (d, p.p.m.) of the O-polysaccharide o f P. mirabilis G1. Additional chemical sh ifts for
NAc are d
H
2.02 and 2.05; d
C
23.6 (2 Me), 175.9 a nd 176.2 (both CO).
Sugar or amino-acid residue H1 H2 H3a, 3b H4 H5 H6a, 6b C1 C2 C3 C4 C5 C6
fi 3)-b-
D
-GalpNAc
I
-(1 fi 4.55 4.02 3.85 4.12 3.70 3.78, 3.78 102.4 52.3 81.3 69.0 76.1 62.4
fi 6)-b-
D
-GalpNAc
II
-(1 fi 4.51 3.89 3.80 4.03 3.85 3.89, 4.16 102.8 53.2 71.1 75.9 73.4 66.7

4
›
fi 4)-b-
D
-GlcpA-(1 fi 4.53 3.36 3.56 3.73 3.76 105.5 73.7 74.9 81.3 76.9 174.6
a-
D
-GalpA-(1 fi 5.20 3.86 4.05 4.30 4.96 100.9 69.5 70.1 71.0 72.6 172.3
L
-Lys 4.38 1.79, 1.91 1.43 1.68 3.00 177.9 54.3 31.9 23.0 27.4 40.5
Fig. 2. 500-MHz
1
H NMR spectrum of the O-polysaccharide of P. mirabilis G1.
Ó FEBS 2002 O-polysaccharide of Proteus mirabilis G1 (Eur. J. Biochem. 269) 1409
d 100.9/4.03 a nd 102.8/3.73, respectively. The other expected
interresidue correlations were either not observed ( for GlcA
C1) or difficult to interpret unambiguously (for GalA H1
and GlcA C1).
The ROESY and HMBC data were in accordance with
the
13
C NMR chemical shift data and were sufficient for
determination of the full monosaccharide sequence in the
repeating unit. A relatively large effect (> 8 p.p.m.) on C1
of GlcA [25] indicated that GlcA and GalNAc in the
b-1 fi 3-linked d isaccharide fragment have t he same
absolute configuration (in case of their different absolute
configuration the effect on C1 would be about < 5 p.p.m.
[26]). Hence, GlcA has the
D

configuration.
On the basis of the data obtained, it was concluded that
the O-polysaccharide P. mirabilis G1 has the structure
shown in Fig. 3. This structure is similar to that of the
O-polysaccharide of P. mirabilis S1959 and OXK from
serogroup O3 [22,23], the r epeating unit of P. mirabilis G1
differing on ly in the absence of the lateral a-
D
-Glcp residue
(Fig. 3).
Serological studies
Rabbit polyclonal anti-(O-polysaccharide) serum against
P. mirabilis G1 was tested in immunohemolysis with LPS
from the complete s et of Proteus stra ins, including 37 strains
of P. mirabilis and 28 strains of P. vulgaris belonging to 49
Proteus O-serogroups as well as 133 strains of P. penneri.
From 188 tested LPS, anti-(O-polysaccharide) serum
against P. mirabilis G1 reacted only with the homologous
LPS and LPS of P. mirabilis S1959, O28, and a mutant of
S1959 (R14, T-like form).
In enzyme immunosorbent assay (EIA), P. mirabilis G1
and P. mirabilis S1959 anti-(O-polysaccharide) sera showed
the strongest reaction w ith LPS of both P. mirabilis G1 and
S1959, whereas LPS of P. mirabilis O28 and R14 reacted
markedly weaker (Fig. 4). The specificity of the cross-
reactions was confirmed b y i nhibition of the reaction in EIA
Fig. 3. Structures of the O-polysaccharides of the cross-reactive LPS of
P. mirabilis G1, S1959, and O28.
Fig. 4. Reactivity of anti-(O-polysaccharide) sera against P. mirabilis
G1 (A) and S1959 (B) in EIA. j,LPSofP. mirabilis G1; d,LPSof

P. mirabilis S195 9; h,LPSofP. mirabilis O 28; s,LPSofP. mirabilis
R14. Antigen do se is 50 ng.
Table 2. Reactivity of absorbed anti-(O-polysaccharide) sera against P. mirabilis G1andS1959withalkali-treatedP. mirabilis LPS in E IA . Sheep
red blood cells were used as control.
Origin of alkali-treated LPS
Reciprocal titre for alkali-treated LPS from
P. mirabilis S1959 P. mirabilis O28 P. mirabilis R14 P. mirabilis G1
P. mirabilis G1 anti-(O-polysaccharide) serum
Control 512 000 256 000 32 000 32 000
P. mirabilis G1 <1000 <1000 <1000 <1000
P. mirabilis S1959 4000 <1000 <1000 <1000
P. mirabilis O28 32 000 32 000 <1000 <1000
P. mirabilis R14 32 000 32 000 <1000 <1000
P. mirabilis S1959 anti-(O-polysaccharide) serum
Control 256 000 256 000 64 000 64 000
P. mirabilis G1 2000 <1000 <1000 <1000
P. mirabilis S1959 <1000 <1000 <1000 <1000
P. mirabilis O28 32 000 32 000 <1000 <1000
P. mirabilis R14 32 000 32 000 <1000 <1000
1410 Z. Sidorczyk et al. (Eur. J. Biochem. 269) Ó FEBS 2002
in the homologous systems of P. mirabilis G1 anti-(O-
polysaccharide) serum/P. mirabilis G1 LPS and P. mirabilis
S1959 anti-(O-polysaccharide) serum/P. mirabilis S1959
LPS. As little as 4–8 ng of the LPS of P. mirabilis G1 and
S1959 were sufficient to inhibit the reaction in both test
systems, whereas two other cross-reactive LPS were signi-
ficantly weaker inhibitors (minimal inhibitory dose 125–
250 ng).
The reactivity of P. mirabilis G1 and S1959 anti-
(O-polysaccharide) sera in EIA was completely abolished

when antisera were absorbed with the homologous LPS
(Table 2). Absorption of P. mirabilis G1 anti-(O-polysac-
charide) serum with P. mirabilis S1959 LPS significantly
decreased the serum titre in the homologous system and
completely removed all cross-reactive antibodies against
LPS of P. mirabilis S1959, R14 and O28. Absorption with
LPS from each of two last strains decreased the reactivity
level with L PS of P. mirabilis G1 a nd S 1959 and c ompletely
abolished the reactivity with LPS of P. mirabilis R14 and
O28. Similar results were obtained with P. mirabilis S1959
anti-(O-polysaccharide) serum absorbed with P. mirabilis
G1 LPS (Table 2).
These results suggested the presence in P. mirabilis G1
and S1959 anti-(O-polysaccharide) sera of cross-reactive
antibodies of at least two types. Antibodies of the first type
bound to an epitope on LPS of P. mirabilis G1 and S1959.
Antibodies of the other type bound to another epitope
shared by the homologous LPS and LPS of all cross-
reactive strains.
In Western b lot, P. mirabilis G1 anti-(O-polysaccharide)
serum recognized slow migrating bands of three LPS
(without R14) and fast migrating bands of P. mirabilis
G1, O 28, and R14 LPS (Fig. 5A). These bands correspond
to high- and low-molecular-mass LPS species consisting of
the core-lipid A moiety with or without an O-chain
polysaccharide attached, respectively. The lack of reactivity
of fast migrating b ands of P. mirabilis S1959 LPS indicated
a difference between the core structures in P. mirabilis
S1959 and G1. Anti-(O-polysaccharide) serum against
P. mirabilis S1959 recognized fast migrating bands of all

LPS tested and slow migrating bands of P. mirabilis S1959,
G1, and O28 LPS, the banding patterns being clearly
different (Fig. 5B). These findings together showed that the
epitope shared by P. mirabilis G1 and S1959 is located in
the O -chain polysacchar ide, whereas epitopes shared by two
other cross-reactive strains are e xposed either on the core or
both on the core an d the O-polysaccharide p art of LPS.
The serological relatedness of P. mirabilis G1 and S 1959
correlated with the similarity of the chemical structures of
their O-polysaccharides (Fig. 3). Therefore, it is reasonable
to classify P. mirabilis G1 into the same serogroup O3 as
P. mirabilis S1959 [22] and to divide this s erogroup into two
subgroups, O 3a,3b for strains P. mirabilis S1959 and OXK
[22] and O3a,3c for strain P. mirabilis G1. The major,
common epitope O3a is a ssociated with a common structure
present in the O-polysaccharides of both strains, which,
most likely, includes a lateral N
a
-(
D
-galacturonoyl)-
L
-lysine
[a-
D
-GalpA6(
L
-Lys)] residue. Previously, this component
was found to play an important role in manifesting the
immunospecificity of P. mirabilis LPS antigens [21,27,28],

including LPS of P. mirabilis S1959 [28]. A partial epitope
O3b is evidently linked t o a lateral a-
D
-Glcp residue which is
present in P. mirabilis S1959 but absent from P. mirabilis
G1, and a p artial epitope O3c in P. mirabilis G1 may b e an
extended epitope that is masked by the a-
D
-Glcp residue in
P. mirabilis S1959.
Comparison of the structures of t he O-chain polysaccha-
rides a nd core oligosaccharides [21–23,27,29,30] enabled
suggestion that
D
-GalpA6(
L
-Lys) is responsible for the
cross-reactivity of not only P. mirabilis G1 and S1959 but
also P. mirabilis O28 and R14. Indeed, LPS of P. mirabilis
O28 is characterized by the presence of a-
D
-GalpA6(
L
-Lys)
in the O-chain polysaccharide [21] (Fig. 3) and
b-
D
-GalpA6(
L
-Lys) in the core oligosaccharide [29]. No

GalA6Lys is present in the polysac charide c hain (T-antigen)
of P. mirabilis R14 LPS [27] but in the LPS core region [27].
However, the level of serological c ross-reactivity of LPS of
Fig. 5. Western blot of LPS of P. mirabilis G1, S1959, R14, and O 28
with anti-(O-polysaccharide) sera against P. mirabilis G1 (A) and S1959
(B).
Ó FEBS 2002 O-polysaccharide of Proteus mirabilis G1 (Eur. J. Biochem. 269) 1411
P. mirabilis O28 and R14 was much lower compared t o t hat
of P. mirabilis G1 and S1959 and the structures of their
O-polysaccharides are significantly different [21,27]. There-
fore, i n spite of the occurrence of t he common LPS epitopes,
these strains should be c lassified separately.
ACKNOWLEDGEMENTS
This work was supported by the Russian F oundation for Basic
Research (grant 99-04-48279) and by the University of Lodz (grant
801).
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