Báo cáo Y học: Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72 pptx

Structural and serological relatedness of the O-antigens
of
Proteus penneri
1 and 4 from a novel
Proteus
serogroup O72
Zygmunt Sidorczyk
1
, Filip V. Toukach
2
, Krystyna Zych
1
, Dominika Drzewiecka
1
, Nikolay P. Arbatsky
2
,
Alexander S. Shashkov
2
and Yuriy A. Knirel
2
1
Department of General Microbiology, Institute of Microbiology and Immunology, University of èodz
Â
, Poland;
2
N. D. Zelinsky
Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
O-speci®c polysaccharides (O-antigens) of the lipopolysac-
charides (LPS) of Proteus penneri strains 1 and 4 were
studied using sugar analysis,

1
Hand
13
C NMR spectroscopy,
including 2D COSY, H-detected
1
H,
13
CHMQC,and
rotating-frame N OE s pectroscop y ( ROESY). The following
structures of the t etrasaccharide (strain 1 ) and pentasac-
charide (strain 4) repeating units of the polysaccharides were
established:
In the polysaccharide of P. penneri strain 4, glycosylation
with the lateral Glc residue ( 75%) a nd 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-speci®c polysaccha-
rides was consistent with the close serological relatedness of
the LPS, which was demonstrated b y immunochemical
studies with O-an tisera against P. penneri 1and4.Basedon
these data, it was proposed to classify P. penneri strains 1
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-speci®c poly-

saccharide; O-serogroup; lipopolysaccharide.
1
Gram-negative bacteria of the genus Proteus are common in
human and animal intestines but under favourable con di-
tions they cause infections of wou nds, burns, skin, eyes,
ears, nose and throat, as well a s intestinal and urinary tract
infections. Strains of two species, Proteus mirabilis and
Proteus vulgaris, have been classi®ed into 60 O -serogroups
[1,2]. Proteus p enneri is a new species proposed for strains
formerly described as Prot eus v ulgaris biogroup I [3,4].
In our immunochemical studies of the outer-membrane
lipopolysaccharides (LPS) aiming at creation of a classi®-
cation scheme for P. penneri, we have found that their
O-speci®c polysaccharide chains are acidic or, less c ommon,
neutral polymers composed of tri- to hexa-saccharide
repeating units [5,6]. As a result of the chemical and
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-speci®c polysaccharides from P. penneri strains 1
and 4 and p ropose 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 èo
Â
dz (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 d escribed previously [8].
6)- -
D
-Glcp-(1 3)- -
D
-GalpNAc-(1 4)- -
D
-Galp-(1
3

1
-
D
-GalpNAc
6)- -
D
-Glcp-(1 3)- -
D
-GalpNAc-(1 4)- -
D
-Galp-(1
6 3

1 1
-
D
-Glcp -
D
-GalpNAc
6
OAc
Correspondence to Z. Sidorczyk, Department of General
Microbiology, Institute of Microbiology and Immunology, University
of Lodz, Banacha 12/16, 90-237 èodz , Poland.
Fax: + 48 42 784932, E-mail:
Abbreviations: LPS, lipopolysaccharide; ROESY, rotating-frame
NOE spectroscopy.
(Received 31 July 2001, revised 17 October 2001, accepted 7 November
2001)
Eur. J. Biochem. 269, 358±363 (2002) Ó FEBS 2002
Isolation of the LPS and O-speci®c polysaccharides
Lipopolysaccharides of P. penneri 1and4wereisolatedin
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
3
CO
2
H a s d escribed previously [10].
Degradation o f 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 ´ 65 cm) of Sephadex G-50 using
0.05
M
pyridinium acetate buffer pH 4.5 as eluent to give
the corresponding O-speci®c 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 1and4
according to a published p rocedure [11].
Agglutination and precipitation tests, SDS/PAGE, elec-
trotransfer of L PS 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 lg) of alkali-treated LPS [12].
Chemical methods
The polysaccharides were hydrolysed with 2
M
CF
3
CO
2
H
(100 °C, 4 h). Amino sugars were identi®ed 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 c olumn of a Dionex Ax8-11 anion-exchange resin in
0.5
M
sodium borate buffer pH 8.0 at 65 °C. The absolute
con®gurations of monosaccharides were determined by
GLC of a cetylated (S)-2-butyl glycosides [14±16] on an
Ultra 2 column using a Hewlett-Packard 5890 chromato-
graph and a temperature program 150±290 °Cat
10 °Cámin
)1
.
O-deacetylation of the strain 4 polysaccharide was
performed with aqueous 12% ammonia at 60 °Cfor2h,
the modi®ed polysaccharide was isolated by gel-permeation
chromatography as described above.
NMR spectroscopy
1
Hand
13
C NMR spectra were recorded with Bruker
AM-300 and Bruker DRX-500 spectrometers in D
2
Oat
60 °C using internal acetone (d
H
2.225, d
C
31.45) as 2D

spectra were obtained u sing standard Bruker s oftware, 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-speci®c polysaccharide
from
P. penneri
strain 1
Sugar analysis of the polysaccharide f rom P. penneri 1
revealed glucose and galactose in almost equal amounts as
well as 2-amino-2-deoxygalactose. All sugars were assigned
to the
D
series using GLC of the acetylated (S)-2-butyl
glycosides [14±16].
The
13
C NMR spectrum of the polysaccharide (Fig. 1)
contained signals for four anomeric carbons at d 99.7±105.6,
two carbons bearing nitrogen at d 52.5 and 54.1, three
nonsubstituted CH
2
OH groups at d 61.9±62.5 and one
O-substituted group at d 66.8(C6ofhexoses;dataofthe
attached-proton test [17]), other sugar ring carbons in the
region d 68.8±81.6, and two N-acetyl groups at d 23.7 (CH
3
)

and 176.0 (CO). Accordingly, the
1
H NMR spectrum
contained, inter alia, signals for four anomeric protons at
d 4.59±4.96 and two N-acetyl groups at d 2.05 and 2.06.
Therefore, the polysaccharide has a tetrasaccharide
repeating unit containing one residue each of
D
-Glc and
D
-Gal and two
D
-residues of GalNAc.
The
1
H- and
13
C NMR spectra of the polysaccharide
were assigned using 2D COSY, H,H-relayed COSY, and
H-detected
1
H,
13
CHMQCexperiments(Tables1and2).
Signals for H1±H4 of each monosaccharide were assigned
directly from the 2D spectra. Signals for H5 of b-linked
sugars (Glcp,GalpNAc
I
and GalpNAc
II

, J
1,2
 8Hz)and
that of a-Galp (J
1,2
4 Hz) were recognized by H1/H5 and
H4/H5 correlations, respectively, which were shown by a
2D ROESY experiment. Signals for H6a,6b of Glc were
assigned on the basis of the H6/C6 correlations, which were
observed in the H-detected
1
H,
13
C HMQC spectrum. The
spin system of Glc was distinguished from those of Gal and
GalNAc on the basis of the
3
J
H,H
coupling constants values
[18], and the spin systems of two GalNAc residues were
Fig. 1. 125-MHz
13
C NMR spectrum of the
O-speci®c polysaccharide of P. penneri 1. The
region of CO resonances is not shown.
Ó FEBS 2002 O-antigens of Proteus penneri 1 and 4 (Eur. J. Biochem. 269) 359
distinguished from t hat of Gal by lower-®eld positions of
the signals for H2 (d 4.07 and 4.02, as compared to d 3.76,
respectively) and by their correlation to the corresponding

nitrogen-bearing carbons at d 52.5 and 54.1. The values
1
J
C1,H1
162.5±165.4 Hz determined from a nondecoupled
1
H,
13
C HMQC spectrum con®rmed the b con®gu ration of
Glc and both GalNAc residues, whereas
1
J
C1,H1
171.1
con®rmed the a con®guration of the Gal residue [19].
Signi®cant low-®eld displacements of the signals for C6 of
Glc, C3 and C4 of Gal, and C3 of GalNAc
I
to d 66.8, 81.3,
77.1, and 81.6 in the
13
C NMR spectrum of the polysac-
charide, compared with their positions in the spectra of the
corresponding u nsubstituted monosaccharides at d 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
GalNA c
I
3-substituted. No signi®cant displacements were

observed for C2-C6 of GalNAc
II
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
H1/Gl c H6b at d 4.95/3.75, Glc H1/GalNAc
I
H3 at
d 4.59/3.89, GalNAc
I
H1/Gal H4 at d 4.96/4.39, and
GalNA c
II
H1/Gal H3 at d 4.61/3.96. These data ®tted
well with the substitution pattern of the sugar residues
determined by the
13
C NMR chemical shift data and
de®ned 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-speci®c polysaccharide of P. penneri 1
has structure 1.
6)- -
D
-Glcp-(1 3)- -
D
-GalpNAc
I

-(1 4)- -
D
-Galp-(1 1
3

1
-
D
-GalpNAc
II
Structure of the O-speci®c polysaccharide
from
P. penneri
strain 4
Sugar analysis of the polysaccharide from P. penneri 4
showed the presence o f the same monosaccharides as in the
polysaccharide from P. penneri 1 but the r elative content of
D
-glucose was twice as high.
Table 2 .
13
C NMR data (d, p.p.m.). Chemical shifts for NAc groups are d 23.7 (CH
3
) and 176.0 (CO).
Sugar residue C1 C2 C3 C4 C5 C6
O-speci®c polysaccharide of P. penneri 1
® 6)-b-
D
-Glcp-(1 ® 105.6 74.3 76.9 70.4 75.4 66.8
® 3)-b-

D
-GalpNAc
I
-(1 ® 102.6 52.5 81.6 69.2 75.7 61.9
® 4)-a-
D
-Galp-(1 ® 99.7 68.8 81.3 77.1 71.5 62.2
3

b-
D
-GalpNAc
II
-(1 ® 105.1 54.1 72.5 69.1 76.2 62.5
O-deacetylated polysaccharide of P. penneri 4
® 6)-b-
D
-Glcp
I
-(1 ® 105.6 74.3 77.0 70.3 75.5 66.6
® 3,6)-b-
D
-GalpNAc
I
-(1 ® 102.6 52.4 81.4 69.4 73.7 68.1
® 4)-a-
D
-Galp-(1 ® 99.8 68.9 81.1 77.6 71.7 62.5
3


b-
D
-GalpNAc
II
-(1 ® 105.0 54.1 72.5 69.1 76.2 62.6
a-
D
-Glcp
II
-(1 ® 99.8 72.6 74.5 71.0 73.3 62.0
Table 1 .
1
HNMRdata(d, p.p.m.). Chemical shifts for NAc groups are d 2.05 and 2.06.
Sugar residue H1 H2 H3 H4 H5 H6a, H6b
O-speci®c polysaccharide of P. penneri 1
® 6)-b-
D
-Glcp-(1 ® 4.59 3.31 3.51 3.55 3.59 4.05, 3.75
® 3)-b-
D
-GalpNAc
I
-(1 ® 4.96 4.07 3.89 4.12 3.66
a
® 4)-a-
D
-Galp-(1 ® 4.95 3.76 3.96 4.39 3.96
a
3


b-
D
-GalpNAc
II
-(1 ® 4.61 4.02 3.74 3.99 3.69
a
O-deacetylated polysaccharide of P. penneri 4
® 6)-b-
D
-Glcp
I
-(1 ® 4.62 3.32 3.51 3.62 3.60 4.05, 3.75
® 3,6)-b-
D
-GalpNAc
I
-(1 ® 4.99 4.09 3.92 4.16 3.87 3.95, 3.73
® 4)-a-
D
-Galp-(1 ® 4.97 3.76 3.95 4.36 3.97
a
3

b-D-GalpNAc
II
-(1 ® 4.61 4.03 3.74 4.00 3.69
a
a-
D
-Glcp

II
-(1 ® 4.96 3.58 3.70 3.44 3.66 3.87, 3.77
a
Signals for H6a and H6b are in the region d 3.65±3.85.
360 Z. Sidorczyk et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The
13
C (Fig. 2) and
1
H NMR spectra of the polysac-
charide demonstrated a structural heterogeneity, most
likely, owing to nonstoichiometric O-acetylation [there
were signals for CH
3
of O-acetyl groups at d
H
2.14 and
2.15, d
C
21.7 (major), 21.4 and 21.5 (both minor)]. After
O-deacetylation with aqueous ammonia, the spectra showed
a higher degree of regularity bu t a number of minor signals
were still present. Assignment of the major series in the
1
H
and
13
C NMR spectra of the O-deacetylated polysaccharid e
using 2D COSY and
1

H,
13
CHMQCexperiments(Tables1
and 2) revealed the same linkage pattern and sugar sequence
as in the polysaccharide of P. penneri 1 (structure 1) and one
additional sugar residue (a-Glcp
II
) attached a t position 6 of
GalNAc
I
.Thea-linkage of Glc
II
followed from the chemical
shifts for C2-C5, w hich were close t o those for a-glucopyr-
anose [20]. The s ite of attachment o f this monosaccharide
was established by displacements of the signals for C5 and
C6 of GalNAc
I
to d 73.7 and 68.1, compared with their
positions at d 75.7 and 61.9, respectively, in the spectrum of
the P. penneri 1 polysaccharide, which are typical of
glycosylation by an a-linked monosaccharide at position 6
[20]. Accordingly, in the
1
HNMRspectrummarked
displacements were observed for the s ignals of GalNAc
I
(in particular, the signal for H5 shifted from d 3.67 to d
3.87), whereas the positions of signals for the other sugar
residues were essentially the same.

Therefore, the m ajor repeating unit of the O-deacetylated
polysaccharide from P. penneri 4 is a pentasaccharide
having structure 2.
6)- -
D
-Glcp
I
-(1 3)- -
D
-GalpNAc
I
-(1 4)- -
D
-Galp-(1 2
6 3

1 1
-
D
-Glcp
II
-
D
-GalpNAc
II
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 lac king Glc
II

and, hence,
having the structure 1. In particular, the signal for C5 of
GalNAc
I
in the minor series had the same chemical shift,
d 75.7, as in the spectrum of the P. penneri 1 polysaccharide
(the signal for C6 of this residue could not be clearly
observed o wing to a coincidence with the signal for C6 of
Glc
II
at d 62.0). Therefore, in the P. penneri 4 polysacchar-
ide the Glc
II
residue is present in a nonstoichiometric
amount. As judged by the ratio of the integral intensities of
the signals in the major and minor series, the average degree
of glycosylation with Glc
II
is  75%.
Comparison of the
13
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
II
are shifted from d 76.2 and 62.6 to d 73.6 and 65.0,
respectively. Such displacements are characteristic for the
effects o f O-acetylation of this monosaccharide a t position 6
[21]. This conclusion was con®rmed by higher-®eld positions

of the signals for H6a and H6b of GalNAc
II
at d 3.70±3.85 in
the
1
H NMR spectrum of the O-deacetylated polysaccharide
compared to those at d 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 in tegral intensities of the signals for the O-acetyl
and N-acetyl groups in the
1
H NMR spectrum, the total
content o f the O-acetyl groups in the repeating unit is 0.75.
In conclusion, the repeating unit of the O-speci®c
polysaccharide of P. penneri 4hasastructuresimilarto
that of P. penneri 1 and differs in the presence of the second
side chain of an a-
D
-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 a nd O-acetylation are
postpolymerization modi®cations [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 t est 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 1
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 im munosorbent ass ay
(Table 3). In all tests, the reactivity of P. penneri 1
O-antiserum with the LPS of P. penn eri 1and4wasalmost
identical, whereas P. penneri 4 O-antiserum reacted with the
LPS of P. penneri 1 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 1
O-antiserum and no cross-reactivity with P. penneri 4
Fig. 2. 125-MHz
13
C NMR spectrum of the
O-speci®c polysaccharide of P. penneri 4.
The region of CO resonances is not show n.
Ó FEBS 2002 O-antigens of Proteus penneri 1 and 4 (Eur. J. Biochem. 269) 361
O-antiserum. The speci®city of the cross-reactions was
con®rmed by p recipitation test and in hibition 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 1 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 1 O-antiserum with all tested antigens was
completely abolished when it was abso rbed with either the

homologous alkali-treated LPS or that of P. penneri 4.
In contrast, absorption of P. pe nneri 4 O-antiserum with the
alkali-treated LPS of P. penneri 1 removed all antibodies to
P. penneri 1 but only a part o f antibo dies to P. penneri 4.
Absorption of P. penneri 1 O-antiserum with the alkali-
treated LPS of P. penneri 40 and 41 abolished binding of
these antigens and signi®cantly decreased binding of the
antigens of P. penneri 1and4.
In Western b lot analysis (Fig. 3), P. penneri 1O-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-speci®c polysac-
charide attached, respectively. Antibodies to P. penneri 1
bound also to low-molecular-mass LPS s pecies of P. penneri
40 and 41. Absorpion of P. penneri 1 O-antiserum with the
P. penneri 40 LPS abolished binding to low-molec ular-mass
LPS species of all strains but remained binding to high-
molecula r-mass LPS spec ies of P. penneri 1and4(datanot
shown). P. penneri 4 O -antiserum clearly r ecognized high-
molecular-mass LPS species of P. penneri 1 and 4 but only
weakly bound to low-molecular-mass LPS species.
These data ®tted well with a marked similarity of the
structures 1 and 2 of the O-speci®c polysaccharides of the
P. penneri 1 and 4 LPS, respectively. The two O-antigens
share the major epitope, wh ich 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
signi®cant 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-antiserum, P. penneri 1 O -antiserum
contained a signi®cant amount o f antibodies to an LPS
core epitope(s), which is shared by all strains studied. Such
Table 3. Reactivit y of O -antis era against P. penneri 1and4withtheLPSofP. pe nneri. LPS and alkali-treated LPS were used as antigen in enzyme
immunosorbent assay and passive immunohemolysis test, respectively. The d ata of the homologous LPS are shown in bold type.
LPS from
P. penneri strain
Reciprocal titre for the LPS in Minimal dose of the LPS in
Enzyme immunosorbent
assay
Passive
immunohemolysis
Precipitation test
(lg)
Inhibition of passive
immunohemolysis (ng)
P. penneri 1 O-antiserum
1 512000 25600 31.7 0.5
4 256000 25600 15.80.5
40 16000 12800 250.07.8
41 16000 12800 250.07.8

P. penneri 4 O-antiserum
1 256000 6400 125.08
4 1024000 51200 7.9 1
40 1000 100 > 1000 > 1000
41 1000 100 > 1000 > 1000
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.
O-antisera absorbed with the
alkali-treated LPS from P. penneri strain
Reciprocal titre with absorbed O-antisera for the alkali-treated LPS from P. penneri strain
144041
P. penneri 1 O-antiserum
Control 25600 25600 12800 12800
1 < 100 < 100 < 100 < 100
4 < 100 < 100 < 100 < 100
40 3200 3200 < 100 < 100
41 3200 3200 < 100 < 100
P. penneri 4 O-antiserum
Control 6400 51200 < 100 < 100
1 < 100 6400 NT NT
4 < 100 < 100 NT NT
362 Z. Sidorczyk et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 immunohemolysis (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
®nding was consistent with the absence of a long-chain
O-speci®c polysaccharide from the LP S of P. penneri 40
(data not shown) and demonstrated the i dentity of t he 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, Polan d) and grant 99 -04-4827 9 from the
Russian Foundation for Basic Research.
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with O-antisera ag ainst P. penneri 1 (A) and 4 (B).
Ó FEBS 2002 O-antigens of Proteus penneri 1 and 4 (Eur. J. Biochem. 269) 363