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,

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


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

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

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

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

-(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-

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


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

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

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

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

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

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Fig. 3. Western blots of the LPS of P. penneri strains 1, 4, 40, and 41
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