Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation O-specific polysaccharides O-antigens of the lipopolysac- charides LPS of Proteus penner
Trang 1Eur J Biochem 269, 358-363 (2002) © FEBS 2002
Structural and serological relatedness of the O-antigens
of Proteus penneri 1 and 4 from a novel Proteus serogroup 072
Zygmunt Sidorczyk’, Filip V Toukach?, Krystyna Zych', Dominika Drzewiecka’, Nikolay P Arbatsky’,
Alexander S Shashkov’ and Yuriy A Knirel?
‘Department of General Microbiology, Institute of Microbiology and Immunology, University of Lodz, Poland; *N D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
O-specific polysaccharides (O-antigens) of the lipopolysac-
charides (LPS) of Proteus penneri strains 1 and 4 were
studied using sugar analysis, 'H and '*C NMR spectroscopy,
including 2D COSY, H-detected 'H,“C HMQC, and
rotating-frame NOE spectroscopy (ROESY) The following
structures of the tetrasaccharide (strain 1) and pentasac-
charide (strain 4) repeating units of the polysaccharides were
established:
—>6)-B-D-Glcp?-(I—>3)-B-D-GalzNAc-(1—>4)-œ-D-Galp?-(I—>
3
†
1 B-D-GalpNAc
—>6)-B-D-Glep-(13)-B-D-GalpNAc-(1>4)-a-D-Galp-(1>
a-D-Glep B-D-GalpNAc
6
|
OAc
In the polysaccharide of P penneri strain 4, glycosylation with the lateral Glc residue (75%) and O-acetylation of the lateral GalNAc residue (55%) are nonstoichiometric This polysaccharide contains also other, minor O-acetyl groups, whose positions were not determined
The structural similarity of the O-specific polysaccha- rides was consistent with the close serological relatedness of the LPS, which was demonstrated by immunochemical studies with O-antisera against P penneri | and 4 Based on these data, it was proposed to classify P penneri strains | and 4 into a new Proteus serogroup, O72, as two sub- groups, O72a and O72a,b, respectively Serological cross- reactivity of P penneri 1 O-antiserum with the LPS of
P penneri 40 and 41 was substantiated by the presence of
an epitope(s) on the LPS core region shared by all
P penneri strains studied
Keywords: Proteus penneri; O-antigen; O-specific poly- saccharide; O-serogroup; lipopolysaccharide
Gram-negative bacteria of the genus Proteus are common in
human and animal intestines but under favourable condi-
tions they cause infections of wounds, burns, skin, eyes,
ears, nose and throat, as well as intestinal and urinary tract
infections Strains of two species, Proteus mirabilis and
Proteus vulgaris, have been classified into 60 O-serogroups
[1,2] Proteus penneri is a new species proposed for strains
formerly described as Proteus vulgaris biogroup I [3,4]
In our immunochemical studies of the outer-membrane
lipopolysaccharides (LPS) aiming at creation of a classifi-
cation scheme for P penneri, we have found that their
O-specific polysaccharide chains are acidic or, less common,
neutral polymers composed of tri- to hexa-saccharide
repeating units [5,6] As a result of the chemical and
Correspondence to Z Sidorczyk, Department of General
Microbiology, Institute of Microbiology and Immunology, University
of Lodz, Banacha 12/16, 90-237 Lodz, Poland
Fax: + 48 42 784932, E-mail: zsidor@biol-uni.lodz.pl
Abbreviations: LPS, lipopolysaccharide; ROESY, rotating-frame
NOE spectroscopy
(Received 31 July 2001, revised 17 October 2001, accepted 7 November
2001)
serological studies of LPS, a number of new Proteus serogroups was proposed for P penneri strains [6,7] Here,
we report on the structures of two neutral structurally related O-specific polysaccharides from P penneri strains | and 4 and propose to classify these strains into a new Proteus serogroup, O72, as two subgroups
MATERIALS AND METHODS
Bacterial strains
P penneri strains 1 (3960-66) and 4 (3266-68) were kindly provided by D J Brenner (Center for Diseases Control, Atlanta, GA, USA) They were isolated from the urine
of patients with bacteriuria and a urinary tract infection
in Michigan and Porto Rico (USA), respectively Further, 66 P penneri strains came from the Collection
of the Department of General Microbiology, University
of Lodz (Poland) Thirty-seven strains of P mirabilis and
28 strains of P vulgaris were from the Czech National Collection of Type Cultures (CNCTC, National Institute
of Public Health, Prague, Czech Republic)
Dry bacterial cells of P penneri 1 and 4 were obtained from aerated cultures as described previously [8]
Trang 2Isolation of the LPS and O-specific polysaccharides
Lipopolysaccharides of P penneri 1 and 4 were isolated in
yields of 4.1 and 9.3% by extraction of bacterial mass with a
hot phenol/water mixture [9] followed by treatment with
cold aqueous 50% CCl,;CO>H as described previously [10]
Degradation of the LPS was performed with aqueous 1%
HOAc at 100 °C for 2 h, a lipid precipitate was removed by
centrifugation (13 000 g, 20 min), and the carbohydrate
portion was fractionated by gel-permeation chromatogra-
phy on a column (3 x 65cm) of Sephadex G-50 using
0.05 M pyridinium acetate buffer pH 4.5 as eluent to give
the corresponding O-specific polysaccharides in a yield of
20% of the LPS mass
Rabbit antisera and serological assays
Polyclonal O-antisera were obtained by immunization of
rabbits with heat-inactivated bacteria of P penneri 1 and 4
according to a published procedure [11]
Agglutination and precipitation tests, SDS/PAGE, elec-
trotransfer of LPS from gels to nitrocellulose sheets, immu-
nostaining, and absorption experiments were carried out as
described in detail previously [12] LPS-BSA complexes were
used as solid-phase antigen in enzyme immunosorbent assay
[13], and passive immunohemolysis was performed with
increasing amounts (2—200 ug) of alkali-treated LPS [12]
Chemical methods
The polysaccharides were hydrolysed with 2 Mm CF;CO.H
(100 °C, 4h) Amino sugars were identified with a
Biotronik LC-2000 amino-acid analyser on a Ostion LG
AN B cation-exchange resin in the standard 0.35 M
sodium citrate buffer pH 5.28 at 80 °C Neutral sugars
were analysed with a Biotronik LC-2000 sugar analyser
on a column of a Dionex Ax8-11 anion-exchange resin in
0.5 M sodium borate buffer pH 8.0 at 65 °C The absolute
configurations of monosaccharides were determined by
GLC of acetylated (S)-2-butyl glycosides [14-16] on an
Ultra 2 column using a Hewlett-Packard 5890 chromato-
graph and a temperature program 150-290 °C at
10 °Cmin `
O-deacetylation of the strain 4 polysaccharide was
performed with aqueous 12% ammonia at 60 °C for 2 h,
the modified polysaccharide was isolated by gel-permeation
chromatography as described above
C1
Fig 1 125-MHz C NMR spectrum of the
O-specific polysaccharide of P penneri 1 The
region of CO resonances is not shown
| tu! a i Pires All a, Ud What 1 \
NMR spectroscopy 'H and °C NMR spectra were recorded with Bruker AM-300 and Bruker DRX-500 spectrometers in DO at
60 °C using internal acetone (64 2.225, dc 31.45) as 2D spectra were obtained using standard Bruker software, and XWINNMR 2.1 program (Bruker) was used to acquire and process NMR data A mixing time of 230 ms was used in a ROESY experiment
RESULTS AND DISCUSSION Structure of the O-specific polysaccharide from P penneri strain 1
Sugar analysis of the polysaccharide from P penneri | revealed glucose and galactose in almost equal amounts as well as 2-amino-2-deoxygalactose All sugars were assigned
to the p series using GLC of the acetylated (S$)-2-butyl glycosides [14-16]
The °C NMR spectrum of the polysaccharide (Fig 1) contained signals for four anomeric carbons at 6 99.7—105.6, two carbons bearing nitrogen at 6 52.5 and 54.1, three nonsubstituted CH;OH groups at 6 61.9-62.5 and one O-substituted group at 6 66.8 (C6 of hexoses; data of the attached-proton test [17]), other sugar ring carbons in the region 6 68.8-81.6, and two N-acetyl groups at 6 23.7 (CHa) and 176.0 (CO) Accordingly, the 'H NMR spectrum contained, inter alia, signals for four anomeric protons at
6 4.59-4.96 and two N-acetyl groups at 6 2.05 and 2.06 Therefore, the polysaccharide has a tetrasaccharide repeating unit containing one residue each of p-Glc and D-Gal and two D-residues of GalNAc
The !H- and '*C NMR spectra of the polysaccharide were assigned using 2D COSY, H,H-relayed COSY, and H-detected 'H,'“C HMQC experiments (Tables 1 and 2) Signals for HI—H4 of each monosaccharide were assigned directly from the 2D spectra Signals for H5 of B-linked sugars (Glep, GalpNAc' and GalpNAc", Ji2 ©8 Hz) and that of o-Galp (7¡› 4 Hz) were recognized by H1/H5 and H4/HS correlations, respectively, which were shown by a 2D ROESY experiment Signals for H6a,6b of Gle were assigned on the basis of the H6/C6 correlations, which were observed in the H-detected 'H,'*C HMQC spectrum The spin system of Glc was distinguished from those of Gal and
GalNAc on the basis of the ° #H„H coupling constants values
[18], and the spin systems of two GalNAc residues were
C6
NAc
| GalNAc C2
|
| KT IT
Trang 3
Table 1 'H! NMR data (6, p.p.m.) Chemical shifts for NAc groups are 5 2.05 and 2.06
O-specific polysaccharide of P penneri 1
3
†
O-deacetylated polysaccharide of P penneri 4
3
†
* Signals for H6a and H6b are in the region 5 3.65—-3.85
Table 2 °C NMR data (6, p.p.m.) Chemical shifts for NAc groups are & 23.7 (CH;) and 176.0 (CO)
O-specific polysaccharide of P penneri 1
3
†
O-deacetylated polysaccharide of P penneri 4
3
†
distinguished from that of Gal by lower-field positions of
the signals for H2 (6 4.07 and 4.02, as compared to 6 3.76,
respectively) and by their correlation to the corresponding
nitrogen-bearing carbons at 6 52.5 and 54.1 The values
Joni 162.5-165.4 Hz determined from a nondecoupled
'H,'°C HMQC spectrum confirmed the B configuration of
Glc and both GalNAc residues, whereas Teun 171.1
confirmed the « configuration of the Gal residue [19]
Significant low-field displacements of the signals for C6 of
Glc, C3 and C4 of Gal, and C3 of GalNAc! to 8 66.8, 81.3,
77.1, and 81.6 in the '*C NMR spectrum of the polysac-
charide, compared with their positions in the spectra of the
corresponding unsubstituted monosaccharides at 6 61.9,
70.4, 70.6, and 72.4 [20], respectively, were due to the effects
of glycosylation and showed that the polysaccharide is
branched, Glc is 6-substituted, Gal 3,4-disubstituted, and
GalNAc! 3-substituted No significant displacements were
observed for C2-C6 of GalNAc" and, hence, it occupies the
terminal position in the side chain
A ROESY experiment revealed the following correla-
tions between the anomeric and linkage protons: Gal
HI/Glc Hób at 8 4.95/3.75, Gle HI/GalNAc' H3 at
5 4.59/3.89, GalNAc' H1/Gal H4 at & 4.96/4.39, and GalNAc" H1/Gal H3 at 5 4.61/3.96 These data fitted well with the substitution pattern of the sugar residues determined by the °C NMR chemical shift data and defined the full sequence of the monosaccharide residues
in the repeating unit
Therefore, on the basis of the data obtained, it was
concluded that the O-specific polysaccharide of P penneri | has structure 1
—6)-B-D-Glep-(13)-B-D-GalpNAc'-(1—4)-a-D-Galp-(l>
3
t
1 B-D-GalpNAc"
Structure of the O-specific polysaccharide from P penneri strain 4
Sugar analysis of the polysaccharide from P penneri 4 showed the presence of the same monosaccharides as in the polysaccharide from P penneri | but the relative content of D-glucose was twice as high
Trang 4C1
Fig 2 125-MHz !C NMR spectrum of the vơ Ne VÝ
O-specific polysaccharide of P penneri 4
The region of CO resonances is not shown
The °C (Fig 2) and 'H NMR spectra of the polysac-
charide demonstrated a structural heterogeneity, most
likely, owing to nonstoichiometric O-acetylation [there
were signals for CH; of O-acetyl groups at dy 2.14 and
2.15, d¢ 21.7 (major), 21.4 and 21.5 (both minor)] After
O-deacetylation with aqueous ammonia, the spectra showed
a higher degree of regularity but a number of minor signals
were still present Assignment of the major series in the 'H
and '°C NMR spectra of the O-deacetylated polysaccharide
using 2D COSY and 'H, °C HMQC experiments (Tables 1
and 2) revealed the same linkage pattern and sugar sequence
as in the polysaccharide of P penneri | (structure 1) and one
additional sugar residue (a-Glep") attached at position 6 of
GalNAc! The o-linkage of Glc' followed from the chemical
shifts for C2-C5, which were close to those for a-glucopyr-
anose [20] The site of attachment of this monosaccharide
was established by displacements of the signals for C5 and
C6 of GalNAc' to 8 73.7 and 68.1, compared with their
positions at 6 75.7 and 61.9, respectively, in the spectrum of
the P penneri 1 polysaccharide, which are typical of
glycosylation by an o-linked monosaccharide at position 6
[20] Accordingly, in the 'H NMR spectrum marked
displacements were observed for the signals of GalNAc!
(in particular, the signal for H5 shifted from 6 3.67 to 6
3.87), whereas the positions of signals for the other sugar
residues were essentially the same
Therefore, the major repeating unit of the O-deacetylated
polysaccharide from P penneri 4 is a pentasaccharide
having structure 2
—>6)-B-D-Glep'-(1—>3)-B-b-GalzNAc'-(1—>4)-œ-D-Galp-(1—> 2
a-D-Glep" B-D-GalpNAc"
A minor series of signals in the NMR spectra of the
O-deacetylated polysaccharide from P penneri 4 resembled
the spectra of the P penneri 1 polysaccharide and belonged
to a tetrasaccharide repeating unit lacking Glc" and, hence,
having the structure | In particular, the signal for C5 of
GalNAc' in the minor series had the same chemical shift,
6 75.7, as in the spectrum of the P penneri 1 polysaccharide
(the signal for C6 of this residue could not be clearly
observed owing to a coincidence with the signal for C6 of
GIc” at ö 62.0) Therefore, in the P penneri 4 polysacchar-
ide the Glc" residue is present in a nonstoichiometric
amount As judged by the ratio of the integral intensities of
DAN ay ermine re eye v
C6
NAc GalNAc C2
(| [ 1
TC LAN Ng r NgV/ÁA v20 Q1, 0.71 1.1.) inal
Chemical shift (p.p.m
the signals in the major and minor series, the average degree
of glycosylation with Glc" is = 75%
Comparison of the °C NMR spectra of the initial and O-deacetylated polysaccharides from P penneri 4 showed that in the former a part of the signals for C5 and C6 of
GalNAc" are shifted from 5 76.2 and 62.6 to ö 73.6 and 65.0,
respectively Such displacements are characteristic for the effects of O-acetylation of this monosaccharide at position 6 [21] This conclusion was confirmed by higher-field positions
of the signals for H6a and H6b of GalNAc" at 8 3.70-3.85 in the 'H NMR spectrum of the O-deacetylated polysaccharide compared to those at 6 4.70 and 4.62 in the spectrum of the initial polysaccharide (a deshielding effect of the O-acetyl group) The average degree of O-acetylation at this position
was estimated as + 55% The sites of attachment of other,
minor O-acetyl groups were not determined Judging from the ratio of integral intensities of the signals for the O-acetyl and N-acetyl groups in the 'H NMR spectrum, the total content of the O-acetyl groups in the repeating unit is 0.75
In conclusion, the repeating unit of the O-specific polysaccharide of P penneri 4 has a structure similar to that of P penneri | and differs in the presence of the second side chain of an o-p-glucopyranose residue and O-acetyl groups The additional substituents occur in nonstoichio- metric amounts, thus indicating that in biosynthesis of the
P penneri 4 O-antigen glucosylation and O-acetylation are
postpolymerization modifications [22]
Serological studies Lipopolysaccharides from 38 strains of P penneri and 65 strains from 49 O-serogroups of P mirabilis and P vulgaris were tested in agglutination test with rabbit polyclonal O-antisera against P penneri 1 and 4 Only three strains,
P penneri 4, 40, and 41, cross-reacted with P penneri |
O-antiserum and only one strain, P penneri 1, with
P penneri 4 O-antiserum
LPS, alkali-treated LPS, and LPS—BSA complexes were
obtained from the cross-reactive strains and tested in passive immunohemolysis and enzyme immunosorbent assay (Table 3) In all tests, the reactivity of P penneri Ì O-antiserum with the LPS of P penneri 1 and 4 was almost identical, whereas P penneri 4 O-antiserum reacted with the LPS of P penneri | more weakly than with the homologous LPS The LPS of P penneri 40 and 41 showed a weak but remarkably similar cross-reactivity with P penneri | O-antiserum and no cross-reactivity with P penneri 4
Trang 5362 Z Sidorezyk et al (Eur J Biochem 269) © FEBS 2002
Table 3 Reactivity of O-antisera against P penneri 1 and 4 with the LPS of P penneri LPS and alkali-treated LPS were used as antigen in enzyme immunosorbent assay and passive immunohemolysis test, respectively The data of the homologous LPS are shown in bold type
P penneri | O-antiserum
P penneri 4 O-antiserum
Table 4 Passive immunohemolysis of the alkali-treated LPS with absorbed O-antisera against P penneri 1 and 4 Sheep red blood cells were used as control NT, not tested
Reciprocal titre with absorbed O-antisera for the alkali-treated LPS from P penneri strain
P penneri | O-antiserum
P penneri 4 O-antiserum
O-antiserum The specificity of the cross-reactions was
confirmed by precipitation test and inhibition of passive
immunohemolysis using various LPS as inhibitors in both
homologous alkali-treated LPS/O-antiserum test systems
(Table 3)
O-Antisera against P penneri | and 4 were absorbed with
the alkali-treated LPS from various strains and tested in
passive immunohemolysis again (Table 4) The reactivity of
P penneri | O-antiserum with all tested antigens was
completely abolished when it was absorbed with either the
homologous alkali-treated LPS or that of P penneri 4
In contrast, absorption of P penneri 4 O-antiserum with the
alkali-treated LPS of P penneri 1 removed all antibodies to
P penneri | but only a part of antibodies to P penneri 4
Absorption of P penneri | O-antiserum with the alkali-
treated LPS of P penneri 40 and 41 abolished binding of
these antigens and significantly decreased binding of the
antigens of P penneri | and 4
In Western blot analysis (Fig 3), P penneri 1 O-antise-
rum reacted with both the slow and fast migrating bands of
the P penneri 1 and 4 LPS, which correspond to high- and
low-molecular-mass LPS species consisting of the core-lipid
A moiety with and without a long-chain O-specific polysac-
charide attached, respectively Antibodies to P penneri 1
bound also to low-molecular-mass LPS species of P penneri
40 and 41 Absorpion of P penneri | O-antiserum with the
P penneri 40 LPS abolished binding to low-molecular-mass LPS species of all strains but remained binding to high- molecular-mass LPS species of P penneri | and 4 (data not shown) P penneri 4 O-antiserum clearly recognized high- molecular-mass LPS species of P penneri | and 4 but only weakly bound to low-molecular-mass LPS species These data fitted well with a marked similarity of the structures | and 2 of the O-specific polysaccharides of the
P penneri | and 4 LPS, respectively The two O-antigens share the major epitope, which was recognized by both O-antisera in all serological tests used, and that of P penneri
4 exposes also a minor epitope, which was bound by
P penneri 4 O-antiserum only and, most likely, is associated with the lateral glucose residue (structure 2) The structures
1 and 2 are unique among Proteus O-antigens, and, accordingly, O-antisera against these strains showed no significant cross-reactivity with O-antigens of any strain from the other known Proteus serogroups Based on these data, we propose to classify P penneri 1 and 4 into a new
Proteus serogroup, O72, as subroups O72a and O72a,72b,
respectively, where a is the major, common epitope and b is
a particular epitope of P penneri 4
Western blot data showed also that, as opposite to
P penneri 4 O-antserum, P penneri 1 O-antiserum contained a significant amount of antibodies to an LPS core epitope(s), which is shared by all strains studied Such
Trang 6A B
¿
Fig 3 Western blots of the LPS of P penneri strains 1, 4, 40, and 41
with Q-antisera against P penneri 1 (A) and 4 (B)
antibodies are present also in P penneri 40 O-antiserum,
whose reactivity with the alkali-treated LPS of P penneri 1,
4, 40 and 41 in passive inmunohemolysis (titres 1 : 25 600,
1: 12 800, 1:25 600 and 1: 25 600, respectively) was
completely abolished by any of the four antigens This
finding was consistent with the absence of a long-chain
O-specific polysaccharide from the LPS of P penneri 40
(data not shown) and demonstrated the identity of the LPS
core epitope(s) in P penneri 1, 4, 40 and 41
ACKNOWLEDGEMENTS
This work was supported by grant 6 P04 A 074 20 from the Sciences
Research Committee (KBN, Poland) and grant 99-04-48279 from the
Russian Foundation for Basic Research
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