Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation The O-chain polysaccharide of the lipopolysaccharide LPS of a previously nonclassified st
Trang 1Structure of the O-polysaccharide and classification
Zygmunt Sidorczyk1, Krystyna Zych1, Filip V Toukach2, Nikolay P Arbatsky2, Agnieszka Zablotni1, Alexander S Shashkov2and Yuriy A Knirel2
1
Department of General Microbiology, Institute of Microbiology and Immunology, University of Lodz, Poland;2N.D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
The O-chain polysaccharide of the lipopolysaccharide (LPS)
of a previously nonclassified strain of Proteus mirabilis
termed G1 was studied by sugar analysis and1H and13C
NMR spectroscopy, including 2D COSY, TOCSY,
rota-ting-frame NOE (ROESY), H-detected1H,13C HMQC, and
heteronuclear multiple-bond correlation (HMBC)
experi-ments The following structure of the polysaccharide was
established:
where D-GalA6(L-Lys) stands for Na-(D
-galacturonoyl)-L-lysine The structure of the O-polysaccharide of
P mirabilis G1 is similar, but not identical, to that of
P mirabilisS1959 and OXK belonging to serogroup O3 Immunochemical studies with P mirabilis G1 and S1959 anti-(O-polysaccharide) sera revealed close LPS-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-ingD-GalA6(L-Lys) in the O-polysaccharide or in the core region
Keywords: Proteus mirabilis; O-polysaccharide; lipopoly-saccharide; Na-(D-galacturonoyl)-L-lysine; serogroup
Much has been written about the taxonomy of Proteus since
the original publication 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 and make up part of the normal flora of
the human gastrointestinal tract Proteus ranks third (after
Escherichiaand 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 with P penneri, it may play a role in some
diarrhoeal diseases [5] Recently, it has been suggested that
P mirabilismay play an ethiopathogenic 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 60 O-serogroups [7,8], including 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 feature 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 groups [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 ofD-galacturonic acid withL-lysine Based on chemical and serological data, we propose to classify this strain in Proteus serogroup O3
M A T E R I A L S A N D M E T H O D S
Bacterial strains and growth
P mirabilisstrains 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 strains 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, Department of General
Microbio-logy, Institute of Microbiology and ImmunoMicrobio-logy, University of Lodz,
90–237, Lodz, Poland Fax and Tel.: + 48 42 635 44 67,
E-mail: zsidor@taxus.biol.uni.lodz.pl
Abbreviations: EIA, enzyme immunosorbent assay; D -GalA(l-Lys),
Na-( D -galacturonoyl)- L -lysine; D -GlcA, D -glucuronic acid; HMBC,
heteronuclear multiple-bond correlation; LPS, lipopolysaccharide;
ROESY, rotating-frame NOE spectroscopy.
(Received 15 October 2001, revised 2 January 2002, accepted
11 January 2002)
Trang 2Institute of Microbiology and Immunology, University of
Lodz, Poland
Dry bacteria were obtained from aerated liquid cultures
as described [14]
Isolation and degradation of lipopolysaccharide
LPS were obtained by extraction of bacterial mass with a
hot phenol/water mixture [15] and purified by treatment
with aqueous 50% CCl3CO2H at 4°C followed by dialysis
of the supernatant Alkali-treated LPS were prepared by
saponification of LPS with 0.25M NaOH (56°C, 2 h)
followed by precipitation with ethanol
Acid degradation of P mirabilis G1 LPS was performed
with 0.1M NaOAc/HOAc buffer pH 4.5 at 100°C for
1.5 h The O-polysaccharide was isolated by gel-permeation
chromatography on a column (3· 65 cm) of Sephadex
G-50 (Pharmacia) using 0.05M pyridinum-acetate buffer
pH 4.5 as eluent; monitoring was performed using a Knauer
differential refractometer (Germany)
Anti-(O-polysaccharide) sera
Rabbit polyclonal anti-(O-polysaccharide) sera against
P mirabilisG1 and P mirabilis S1959 were obtained by
intravenous immunization of rabbits every 5 days with 0.25,
0.5 and 1.0 mL bacterial suspension (1.5· 1010c.f.u.ÆmL)1),
boiled at 100°C for 2 h One week after the last injection,
rabbits were bled The obtained antisera were stored at
)20 °C For passive immunohemolysis, the antisera were
inactivated at 56°C for 30 min and absorbed with sheep red
blood cells One hemolytic unit of anti-(O-polysaccharide)
serum was defined as the antibody dilution yielding 50%
lysis of sheep red blood cells
Agglutination test
Agglutination in tubes was performed with 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 alkali-treated LPS for 30 min at 37°C, then
washed with NaCl/PipH 7.2 (15 mM Na2HPO4, 150 mM
NaCl) and suspended at a concentration 0.5% in veronal
buffer pH 7.3 (1.8 mM sodium 5,5-diethylbarbiturate,
3.1 mM 5,5-diethylbarbituric acid, 150 mM NaCl, 0.5 mM
MgCl2, 0.15 mMCaCl2) Anti-(O-polysaccharide) serum was
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 at 37°C for 1 h The last dilution of
antiserum giving 50% hemolysis was established as a titre
A total of 25 lL anti-(O-polysaccharide) serum
contain-ing 2 or 3 hemolytic units of antibodies was incubated with
25 lL twofold serially diluted inhibitor in microtitrate
plates After incubation (15 min, 37°C), 50 lL sensitized
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 diluted with NaCl/Pi(1 : 50) was treated with 100 lL sheep red blood cells (0.2 mL) sensitized with the respective antigen (200 lg LPS) for 30 min in an ice bath 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/Piat 4°C for 16 h and washed with water Plates were blocked with 2.5% casein in NaCl/Pi(incubation with NaCl/Pi/casein for 1 h at 37°C followed by two washing cycles with NaCl/Pi) and anti-(O-polysaccharide) serum diluted appropriately with NaCl/Pi/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/Pi/casein was added and incubation was continued for 1 h at 37°C After washing in NaCl/Pi, the plates were washed twice in substrate buffer (0.1M 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 lL 0.1%
H2O2was added After incubation for 30 min at 37°C, the reaction was stopped by adding aqueous 2% oxalic acid, and the 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
A405> 0.2
Inhibitor was serially twofold diluted with 30 lL NaCl/
Pi/casein and mixed in V-shaped microtitrate plates (Med-lab, Poland) with an equal volume of antibodies diluted with the same buffer to give A405of 1.0–1.6 without adding the inhibitor After incubation at 37°C for 15 min, the mixture was transferred to EIA plates coated with LPS, and further steps were performed as described above
SDS/PAGE and Western blot SDS/PAGE and Western immunoblots were 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 and then transferred to
a nitrocellulose membrane The membrane was blocked with 10% skimmed milk in dot-blot buffer pH 7.4 (50 mM Tris/HCl and 200 mMNaCl) at 20°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°C for 2 h 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 3M CF3CO2H (100°C, 4 h), amino and neutral sugars were identified using Biotronik LC-2000 amino-acid and sugar analysers as
Trang 3described [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°C at 3 °CÆmin)1
NMR spectroscopy
Samples were deuterium-exchanged by freeze-drying two
times from D2O and examined in D2O at 45°C using
internal acetone as reference (dH2.225, dc31.45).1H and13C
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, andXWINNMRprogram (Bruker)
was used to acquire and process data A mixing time of 200
and 300 ms was used in TOCSY and ROESY experiments,
respectively
R E S U L T S A N D D I S C U S S I O N
Structural studies
The O-polysaccharide was prepared by mild acid
degrada-tion of P mirabilis G1 LPS followed by gel-permeadegrada-tion
chromatography on Sephadex G-50 Sugar analysis of the
polysaccharide after acid hydrolysis revealed glucuronic acid
(GlcA) and galacturonic acid (GalA) in the ratio 1 : 5
Analysis on an amino-acid analyser showed the presence of
2-amino-2-deoxygalactose and lysine TheDconfiguration
of GalA and GalN was determined by GLC of the
acetylated (S)-2-butyl glycosides and theLconfiguration of
lysine by GLC of the acetylated (S)-2-butyl ester TheD
configuration of GlcA was established by analysis of13C
NMR chemical shift data of the polysaccharide (see below)
The13C NMR spectrum of the polysaccharide (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-CH2OH groups (C6 of GalN, data of attached-proton test [20]), two carboxyl groups at d 172.3 and 174.6 (C6 of GlcA and GalA), two carbons bearing nitrogen at d 52.3 and 53.2 (C2 of GalN), 14 sugar ring carbons bearing oxygen in the region d 69.0–81.3, two N-acetyl groups (CH3
at d 23.6, CO at d 175.9 and 176.2), and six carbons of lysine
at d 23.0, 27.4, 31.9, 40.5, 54.3 and 177.9 (Table 1, compare published data [21–23]) Accordingly, the1H NMR spec-trum of the polysaccharide (Fig 2) contained signals 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, the polysaccharide has a tetrasaccharide repeating unit containing one residue each ofD-GlcA and
D-GalA, two residues ofD-GalNAc, andL-lysine A smaller than expected relative content of GlcA in the polysaccharide hydrolysate could be accounted for by its retention in oligosaccharides with GalN (see the polysaccharide struc-ture below)
The1H and13C NMR spectra of the polysaccharide were assigned using 2D COSY, TOCSY, ROESY, 1H,13C 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 (GalNAcIand GalNAcII) The signals for H5 and H6 of GalNAcIwere assigned by H4/H5 correlation in the ROESY spectrum and H5/H6 correlation
in the COSY spectrum The corresponding 13C NMR signals were found by1H,13C correlations in the HMQC spectrum, and three remaining signals were assigned to H5/C5, H6a/C6, and H6b/C6 correlations of GalNAcII
JH,H coupling constant values estimated from the 2D COSY and TOCSY spectra were typical of sugars with the glucoand galacto configurations in the pyranose form [24] The GlcA residue was identified by a large J3,4coupling constant value of 10 Hz, as compared with J3,4 £ 3 Hz for the other sugars that have the galacto configuration The
Fig 1 125-MHz13C NMR spectrum of the O-polysaccharide of P mirabilis G1.
Trang 4GalNAc residues were distinguished by correlation
of protons at carbons bearing nitrogen (H2) to the
corresponding carbons (C2) revealed by the1H,13C HMQC
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 of Lys and C6 of GalA, thus demonstrating the
presence of Na-galacturonoyllysine (GalA6Lys) This
con-clusion was confirmed by typical13C 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 J1,2coupling constant values of 7–8 Hz
determined from the1H 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 the13C NMR chemical shift
data (Table 1, compare to published data [23])
Significant downfield displacements of the signals for C3
of GalNAcI, C4 of GlcA, C4 and C6 of GalNAcIIto 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 13C NMR chemical shifts for the GalA residue were close to those for the nonsubstituted monosaccharide [23] and, hence, this residue is terminal
The ROESY spectrum showed a GalA H1/GalNAcIIH4 correlation at d 5.20/4.03, and, hence, GalALys is attached
to the disubstituted GalNAcIIresidue as a monosaccharide side chain Correlations of the b-linked sugars in the main chain were difficult to interpret because of close positions of the H1 resonances and multiple coincidences of intraresidue H1/H3,H5 and interresidue cross-peaks The HMBC spec-trum contained a GalNAcIH1/GalNAcIIC6 at d 4.55/66.7 and two overlapping cross-peaks at 4.51–4.53/81.3, which could be assigned to GalNAcIIH1/GlcA C4 and GlcA H1/ GalNAcIC3 correlations In addition, GalA C1/GalNAcII H4 and GalNAcIIC1/GlcA H4 cross-peaks were present at
Table 1 500-MHz 1 H and 125-MHz 13 C NMR chemical shifts (d, p.p.m.) of the O-polysaccharide of P mirabilis G1 Additional chemical shifts for NAc are d H 2.02 and 2.05; d C 23.6 (2 Me), 175.9 and 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 -GalpNAcI-(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 -GalpNAcII-(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-MHz1H NMR spectrum of the O-polysaccharide of P mirabilis G1.
Trang 5d 100.9/4.03 and 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
the13C 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-1fi 3-linked disaccharide fragment have the 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 theDconfiguration
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 repeating unit of P mirabilis G1
differing only in the absence of the lateral a-D-Glcp residue
(Fig 3)
Serological studies
Rabbit polyclonal anti-(O-polysaccharide) serum against
P mirabilisG1 was tested in immunohemolysis with LPS
from the complete set of Proteus strains, including 37 strains
of P mirabilis and 28 strains of P vulgaris belonging to 49 ProteusO-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 with 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 by inhibition 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, LPS of P mirabilis G1; d, LPS of
P mirabilis S1959; h, LPS of P mirabilis O28; s, LPS of P mirabilis R14 Antigen dose is 50 ng.
Table 2 Reactivity of absorbed anti-(O-polysaccharide) sera against P mirabilis G1 and S1959 with alkali-treated P mirabilis LPS in EIA 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
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
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
Trang 6in 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 LPS of P mirabilis G1 and S1959 and completely 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 blot, P mirabilis G1 anti-(O-polysaccharide) serum recognized slow migrating bands of three LPS (without R14) and fast migrating bands of P mirabilis G1, O28, 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 bands 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 polysaccharide, whereas epitopes shared by two other cross-reactive strains are exposed either on the core or both on the core and the O-polysaccharide part of LPS The serological relatedness of P mirabilis G1 and S1959 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 mirabilisS1959 [22] and to divide this serogroup into two subgroups, O3a,3b for strains P mirabilis S1959 and OXK [22] and O3a,3c for strain P mirabilis G1 The major, common epitope O3a is associated with a common structure present in the O-polysaccharides of both strains, which, most likely, includes a lateral Na-(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 to a lateral a-D-Glcp residue which is present in P mirabilis S1959 but absent from P mirabilis G1, and a partial epitope O3c in P mirabilis G1 may be an extended epitope that is masked by the a-D-Glcp residue in
P mirabilisS1959
Comparison of the structures of the O-chain polysaccha-rides and core oligosacchapolysaccha-rides [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 polysaccharide chain (T-antigen)
of P mirabilis R14 LPS [27] but in the LPS core region [27] However, the level of serological cross-reactivity of LPS of
Fig 5 Western blot of LPS of P mirabilis G1, S1959, R14, and O28
with anti-(O-polysaccharide) sera against P mirabilis G1 (A) and S1959
(B).
Trang 7P mirabilisO28 and R14 was much lower compared to that
of P mirabilis G1 and S1959 and the structures of their
O-polysaccharides are significantly different [21,27]
There-fore, in spite of the occurrence of the common LPS epitopes,
these strains should be classified separately
A C K N O W L E D G E M E N T S
This work was supported by the Russian Foundation for Basic
Research (grant 99-04-48279) and by the University of Lodz (grant
801).
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