Báo cáo khoa học: Structure of the O-polysaccharide fromProteus myxofaciens Classification of the bacterium into a newProteusO-serogroup pptx

Structure of the O-polysaccharide from
Proteus myxofaciens
Classification of the bacterium into a new
Proteus
O-serogroup
Zygmunt Sidorczyk
1
, Anna N. Kondakova
2
, Krystyna Zych
1
, Sof’ya N. Senchenkova
2
,
Alexander S. Shashkov
2
, Dominika Drzewiecka
1
and Yuriy A. Knirel
2
1
Department of General Microbiology, Institute of Microbiology and Immunology, University of Ło
´
dz
´
, Poland;
2
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
The O-polysaccharide (O-antigen) was obtained from the
lipopolysaccharide of Proteus myxofaciens,aProteus
strain producing copious amounts of slime, which was

isolated from the gypsy moth larvae. The structure of the
polysaccharide was studied by chemical analysis and
1
H
and
13
C NMR spectroscopy, including 2D COSY,
TOCSY, ROESY and H-detected
1
H,
13
CHMQC
experiments. It was found that the polysaccharide contains
an amide of glucuronic acid (GlcA) with an unusual
a-linked amino acid, N
e
-[(R)-1-carboxyethyl]-
L
-lysine
(2S,8R-alaninolysine, 2S,8R-AlaLys), and has a linear
tetrasaccharide repeating unit of the following structure:
This structure is unique among known bacterial poly-
saccharide structures. On the basis of these and sero-
logical data, it is proposed that P. myxofaciens be
classified into a new Proteus serogroup, O60, of which
this strain is the single representative. Structural and
serological relatedness of P. myxofaciens to other AlaLys-
containing O-antigens of Proteus and Providencia is
discussed.
Keywords: lipopolysaccharide; N

e
-[(R)-1-carboxyethyl]-
L
-lysine; O-polysaccharide; O-serogroup; Proteus myxo-
faciens.
Gram-negative bacteria of the genus Proteus from the
family Enterobacteriaceae are divided into four species:
P. vulgaris, P. mirabilis, P. penneri and P. hauseri,aswell
as three unnamed Proteus genomospecies 4, 5 and 6 [1,2].
They are widely distributed in nature and are important
facultative human and animal pathogens, which in
favorable conditions cause mainly intestine and urinary
tract infections that sometimes lead to serious complica-
tions, such as acute or chronic pyelonephritis and
formation of bladder and kidney stones. They may also
be the source of wound, burn, skin, nose, and throat
infections [3]. Recently, it has been suggested that
P. mirabilis plays an etiopathogenic role in rheumatoid
arthritis [4].
Potential virulence factors of Proteus rods, which mediate
infectious processes, are flagella, fimbriae, invasiveness,
enzymes, e.g. proteases and ureases, haemolysins, capsular
polysaccharide and lipopolysaccharide (endotoxin, LPS)
[5,6]. The serological specificity of the bacteria is defined
by the structure of the O-specific polysaccharide chain
(O-antigen) of the lipopolysaccharide. On the basis of the
O-antigens, the strains of two species, P. vulgaris and
P. mirabilis, have been classified into 49 O-serogroups [7]
and later into 11 additional serogroups [8]. About 15 further
O-serogroups have been proposed for the third medically

important species, P. penneri [9,10]. Structures of the
O-polysaccharides of most Proteus serogroups have been
determined and correlated with the immunospecificity of
the O-antigens [9]. Most O-polysaccharides studied so far
(>80%) contain acidic or both acidic and basic compo-
nents, such as uronic acids, their amides with amino acids,
phosphate, ethanolamine phosphate and other nonsugar
constituents [9].
In 1966 another Proteus strain that produced copious
amounts of slime was isolated from living and dead larvae
of the gypsy moth (Porthetria dispar) and called Proteus
myxofaciens [11]. Its medical importance and position in the
serological classification of the genus Proteus remains
unknown.
In this work we report on the structure of the
O-polysaccharide of P. myxofaciens and serological pro-
perties of the LPS of this strain, including cross-reactivity
with several other Proteus and Providencia strains with
Correspondence to Z. Sidorczyk, Department of General
Microbiology, Institute of Microbiology and Immunology,
University of Ło
´
dz´ , Banacha 12/16, 90-237 Ło
´
dz´ , Poland.
Fax: + 48 42 6784932, E-mail:
Abbreviations: EIA, enzyme immunosorbent assay; GlcA, glucuronic
acid; HMQC, heteronuclear multiple-quantum correlation; LPS,
lipopolysaccharide; 2S,8R-AlaLys and 2S,8S-AlaLys, N
e

-[(R)-
and (S)-1-carboxyethyl]-
L
-lysine (2S,8R-alaninolysine and
2S,8S-alaninolysine).
(Received 28 February 2003, revised 5 May 2003,
accepted 2 June 2003)
Eur. J. Biochem. 270, 3182–3188 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03698.x
structurally related O-polysaccharides. Together with the
genera Proteus and Morganella, the bacteria Providencia
comprise the third genus in the tribe Proteeae [12]. Members
are distinguished by common morphological, cultural and
enzymatic properties. Currently, the genus Providencia
consists of five species: Pr. alcalifaciens, Pr. heimbache,
Pr. rettgeri, Pr. rustigianii and Pr. stuartii [1,12]. On the
basis of structural and serological data, we propose the
classification of P. myxofaciens into a new, separate Proteus
serogroup, O6O.
Materials and methods
Bacterial strains and growth
P. myxofaciens strain 18769-CCUG (ATCC 19692) was
kindly provided by E. Falsen [Culture Collection, Univer-
sity of Goeteborg (CCUG), Goeteborg, Sweden]. P. mira-
bilis O13 was from the Collection of the Institute of
Microbiology and Immunology (University of Ło
´
dz´ ).
Pr. alcalifaciens O23 and Pr. rustigianii O14 were from the
Hungarian National Collection of Medical Bacteria
(National Institute of Hygiene, Budapest, Hungary). Dry

bacterial mass was obtained from aerated culture as
described previously [13].
Isolation of the LPS and O-polysaccharide
LPS of P. myxofaciens wasisolatedinayieldof6.23%of
dried cell weight by phenol/water extraction [14] followed
by treatment with cold aq. 50% trichloroacetic acid to
precipitate nucleic acids as described [15]. The LPS was
degraded with 1% (v/v) acetic acid at 100 °Cfor2h.The
lipid precipitate was removed by centrifugation (13 000 g,
20 min), and the carbohydrate portion was fractionated by
gel chromatography on a column (65 · 2.6 cm) of Sepha-
dex G-50 using pyridinum acetate buffer as eluent (10 mL
acetic acid and 4 mL pyridine in 1 L water) to give a high-
molecular-mass O-polysaccharide in a yield of 20% of the
LPS weight.
Rabbit antisera and serological assays
Polyclonal O-antisera were obtained by immunization of
rabbits with heat-inactivated bacteria of P. myxofaciens and
P. mirabilis O13 as described [16]. SDS/PAGE, electroblot-
ting, immunostaining, enzyme immunosorbent assay (EIA),
and absorption experiments were carried out as described
[17]. LPS was used as antigen in the EIA.
Sugar and amino-acid analyses
Polysaccharide was hydrolysed with 2
M
trifluoroacetic acid
(120 °C, 2 h), and the monosaccharides were analysed by
GLC as the acetylated alditols [18]. Amino components
were identified using a Biotronik LC-2000 amino-acid
analyzer on a column (0.4 · 22cm)ofOstionLGANB

cation-exchange resin at 80 °Cin0.2
M
sodium citrate
buffer, pH 3.25, for amino acids and 0.35
M
sodium citrate
buffer, pH 5.28, for amino sugars. Uronic acid was
identified using a Biotronik LC-2000 sugar analyzer on a
Chromex DA · 8–11 column at 70 °Cin0.04
M
KH
2
PO
4
buffer, pH 2.4. The absolute configuration of the amino
sugars was determined by GLC of the acetylated (S)-2-butyl
glycosides as described [19,20]. N
e
-[(R)-1-Carboxyethyl]-
L
-lysine was isolated from the polysaccharide hydrolysate
(2
M
trifluoroacetic acid, 120 °C, 2 h) by gel chromato-
graphy on a column (80 · 1.6 cm) of TSK HW-40 in water.
NMR spectroscopy
1
Hand
13
C NMR spectra were recorded with a Bruker

DRX-500 spectrometer equipped with an SGI INDY
computer workstation using internal acetone as reference
(d
H
2.225, d
C
31.45). 2D NMR spectra were obtained using
standard Bruker software, and the
XWINNMR
2.1 program
(Bruker) was used to acquire and process the NMR data.
A mixing time of 200 and 300 ms was used in TOCSY
and ROESY experiments, respectively.
Results and discussion
Structural studies
LPS was obtained from dried bacterial cells of P. myxofac-
iens and degraded with dilute acetic acid to give a
high-molecular-mass O-polysaccharide isolated by gel-per-
meation chromatography on Sephadex G-50. Analysis of
the polysaccharide using an amino-acid analyzer revealed
the presence of GlcN and GalN in the ratio 2 : 1 as well as
another amino component. Sugar analysis using anion-
exchange chromatography demonstrated the presence of
glucuronic acid (GlcA). The
D
configuration of GlcN and
GalN was established by GLC of the acetylated (S)-2-butyl
glycosides. The
D
configuration of GlcA was determined by

analysis of the
13
C NMR chemical-shift data of the
polysaccharide (see below).
The specific optical rotation values of 2S,8R-AlaLys for
2S,8S-AlaLys, +9.7 and +11.6, are too close to each other
to be useful for differentiation between these two isomers,
especially when the natural sample is not crystalline.
However, the optical rotation value is useful for differen-
tiation between 2S and 2R isomers because both 2S
isomers (2S,8R and 2S,8S) have a positive value (published
data [21]), whereas both 2R isomers (2R,8R and 2R,8S)
would have a corresponding negative value, )9.7 and
)11.6. Therefore, from the specific optical rotation value of
+13 we can infer that the natural sample has the 2S
configuration, i.e. that lysine is
L
as in all AlaLys isomers
found so far in various natural sources. Differentiation
between the 8S and 8R isomers in favor of the latter was
made from the
13
C NMR data as described previously
([22,23]).
1
The
13
C NMR spectrum (Fig. 1) suggested that the
polysaccharide is regular and has a tetrasaccharide repeat-
ing unit. It contained signals for four anomeric carbons at d

98.7–104.0, five nitrogen-bearing carbons at d 47.0–58.8 (C2
of GlcN and GalN, C2 and C6 of AlaLys), one unsubsti-
tuted (d 61.9) and two substituted (d 66.9 and 70.1)
HOCH
2
-C groups (data of attached-proton test), one
CH
3
-C group of AlaLys at d 16.2, three C-CH
2
-C groups
of AlaLys at d 23.4, 26.5 and 31.9, three N-acetyl groups
(CH
3
at d 23.5–23.8), and six carbonyl groups at d 170.1 (C6
of GlcA) and 175.2–177.9 (CO of N-acetyl groups and
Ó FEBS 2003 O-Polysaccharide of P. myxofaciens (Eur. J. Biochem. 270) 3183
COOH of AlaLys). A relatively high-field position of the
signal for C6 of GlcA showed that the uronic acid is
amidated by AlaLys (compare with published data [23,24]).
The
1
H NMR spectrum (Fig. 2) contained signals for four
anomeric protons at d 4.40–4.92, one CH
3
-C group at d
1.47, three C-CH
2
-C groups at d 1.48–1.93, and three
N-acetyl groups at d 2.01–2.10.

These data together suggest that the tetrasaccharide
repeating unit of the polysaccharide consists of two residues
of GlcNAc and one residue each of GalNAc, GlcA and
AlaLys.
The
1
Hand
13
C NMR spectra of the polysaccharide
were assigned using 2D NMR experiments, including
1
H,
1
H COSY, TOCSY, ROESY and H-detected
1
H,
13
C
heteronuclear multiple-quantum correlation (HMQC),
and the results are summarized in Table 1. On the basis
of characteristic coupling constants [25], spin systems of
two b-GlcpNAc residues (GlcNAc
I
and GlcNAc
II
),
a-GalpNAc and b-GlcpA, were identified. In particular,
the configurations of the glycosidic linkages were deter-
mined by the J
1,2

coupling constant values of 7–8 Hz for
the b-linked and  4Hz for the a-linked sugar pyrano-
sides. The remaining, nonsugar signals were assigned to
AlaLys. The signal for H2 of AlaLys was shifted
downfield to d 4.34, as compared with its position near
d 3.8 in the free amino acid [23], indicating its acylation
at N2.
Low-field displacements of the signals for C4 of GlcA
and C3 of b-GlcNAc
II
as well as C6 of b-GlcNAc
I
and
a-GalNAc to d 77.6, 84.1, 66.9 and 70.1, as compared with
their positions in the spectra of the corresponding unsub-
stituted monosaccharides at d 72.9, 74.8, 61.9, and 62.1,
respectively [26], demonstrated the modes of glycosylation
of the monosaccharides.
The linkage positions were confirmed and the sequence of
the monosaccharides was determined using a 2D ROESY
experiment, which revealed the following correlations
between the anomeric protons and protons at the linkage
carbons: GalNAc H1/GlcNAc
I
H6 at d 4.92/3.71 and 4.92/
3.98, GlcNAc
I
H1/GlcpNAc
II
H3 at d 4.62/3.65, GlcNAc

II
H1/GlcA H4 at d 4.40/3.95, and GlcA H1/GalNAc H6 at
d 4.58/3.87 and 4.58/4.01.
In the
13
C NMR spectrum of the polysaccharide, a
relatively large b effect ()2 p.p.m.) was observed on the C3
Fig. 1.
13
C NMR spectrum of the O-polysac-
charide of P. myxofaciens.
Fig. 2.
1
H NMR spectrum of the O-polysac-
charide of P. myxofaciens.
3184 Z. Sidorczyk et al.(Eur. J. Biochem. 270) Ó FEBS 2003
signal of GlcA, which was caused by its glycosylation at
position 4 with b-
D
-GlcNAc and showed that both mono-
saccharides have the same absolute configuration, i.e. that
GlcA has the
D
configuration (the b effect on C3 of
L
-GlcA
wouldbeclosetozero[27]).
On the basis of the data obtained, it was concluded that
the repeating unit of the O-polysaccharide of P. myxofac-
iens has the following structure:

The polysaccharide contains an amide of
D
-glucuronic
acid with an unusual amino acid, N
e
-[(R)-1-carboxyethyl]-
L
-lysine (structure 1 in Fig. 3). The same amide has
previously been identified in the O-polysaccharide of
Pr. alcalifaciens O23 [23], and an amide of the same amino
acid with
D
-galacturonic acid (structure 2) in the O-polysac-
charides of P. mirabilis O13 [24]. An amide of
D
-galacturonic
acid with an isomeric amino acid, N
e
-[(S)-1-carboxyethyl]-
L
-
lysine (structure 3), has been found in the O-polysaccharide
of Pr. rustigianii O14 [28]. Structures of the O-polysac-
charides that contain N
e
-[(R)-1-carboxyethyl]-
L
-lysine and
N
e

-[(S)-1-carboxyethyl]-
L
-lysine are shown in Fig. 4. Some
other O-polysaccharides of Proteus contain amide-linked
L
-lysine or aminoalkyl phosphate groups, which, like
N
e
-(1-carboxyethyl)-
L
-lysine, endow the polysaccharides
with a zwitterionic character.
Serological studies
LPS of a number of Proteus strains with known
O-polysaccharide structure and those from Pr. rustigianii
O14 and Pr. alcalifaciens O23 were tested for serological
relatedness to P. myxofaciens. Only LPS of P. mirabilis O13
and Pr. rustigianii O14, both containing AlaLys [24,28],
reacted strongly with polyclonal rabbit P. myxofaciens
O-antiserum in an EIA, although the cross-reactivity was
weaker than the reactivity of the homologous LPS
(Table 2). P. mirabilis O13 O-antiserum reacted with the
LPS of all three strains. However, the inhibiting doses of
the cross-reactive LPS were significantly higher than that
of the homologous LPS in both P. myxofaciens and
P. mirabilis O13 homologous test systems (Table 2).
Another AlaLys-containing LPS, that from Pr. alcalifaciens
O23 [23], cross-reacted with O-antisera against P. myxofac-
iens and P. mirabilis O13 only weakly (Table 2).
Reactivity in EIA of both O-antisera with all tested LPS

was completely abolished when they were absorbed with the
homologous LPS (Table 3). Absorption of P. myxofaciens
O-antiserum with the LPS of P. mirabilis O13 and Pr. rust-
igianii O14 influenced its reactivity with the homologous
LPS only slightly, whereas a significant decrease in the
reactivity with the homologous LPS was observed when
P. mirabilis O13 O-antiserum was absorbed with the LPS of
P. myxofaciens and Pr. rustigianii O14. All tested LPS
species completely removed cross-reactive antibodies from
both O-antisera (Table 3). Absorption with Pr. alcalifaciens
O23 LPS did not influence the reaction of the O-antisera
with the other LPS species (data not shown).
In Western blot, all tested LPS species showed similar
patterns with O-antisera against P. myxofaciens and P. mir-
abilis O13 with respect to slow moving LPS species with
a long-chain O-polysaccharide (Fig. 5). Therefore, the
cross-reactive LPS species share epitopes on the O-polysac-
charide; the structures are shown in Fig. 4. The strong cross-
reactivity of P. mirabilis O13 O-antiserum with the LPS of
P. myxofaciens and Pr. rustigianii O14 (Table 2) is prob-
ably due to the abundance of antibodies against AlaLys,
which was demonstrated to be of importance in manifesting
Table 1.
1
H and
13
C NMR chemical shifts for the O-polysaccharide of P. myxofaciens.
Sugar residue
Chemical shift (d, p.p.m.)
Sugar atoms

N-Acetyl or 1-carboxyethyl
atoms
H1 H2 H3 H4 H5 H6 H2¢ H3¢
fi 6)-a-
D
-GalpNAc-(1 fi 4.92 4.20 3.97 4.01 4.13 3.87 4.01 2.01
a
fi 6)-b-
D
-GlcpNAc
I
-(1 fi 4.62 3.73 3.58 3.60 3.68 3.71 3.98 2.09
a
fi 3)-b-
D
-GlcpNAc
II
-(1 fi 4.40 3.77 3.65 3.51 3.41 3.73 3.90 2.10
a
fi 4)-b-
D
-GlcpA-(1 fi 4.58 3.36 3.63 3.95 3.97
2S,8R-AlaLys 4.34 1.76 1.93 1.48 1.72 3.06 3.72 1.47
C1 C2 C3 C4 C5 C6 C1¢ C2¢ C3¢
fi 6)-a-
D
-GalpNAc-(1 fi 98.7 50.9 68.5 69.7 71.0 70.1 175.2
a
23.5
b

fi 6)-b-
D
-GlcpNAc
I
-(1 fi 102.7 56.8 74.8 70.7 75.3 66.9 175.6
a
23.5
b
fi 3)-b-
D
-GlcpNAc
II
-(1 fi 100.6 55.2 84.1 70.0 76.7 61.9 175.7
a
23.8
b
fi 4)-b-
D
-GlcpA-(1 fi 104.0 73.7 74.6 77.6 75.4 170.1
2S,8R-AlaLys 177.9 55.7 31.9 23.4 26.5 47.0 175.8
a
58.8 16.2
a,b
Assignment could be interchanged.
Ó FEBS 2003 O-Polysaccharide of P. myxofaciens (Eur. J. Biochem. 270) 3185
the P. mirabilis O13 specificity [29]. The configuration of
neither AlaLys nor uronic acid amidated by AlaLys seems
to play any significant role in the recognition. Indeed,
antibodies against AlaLys could be completely absorbed by
both LPS of Pr. rustigianii O14 containing AlaLys of a

different configuration (2S,8S vs. 2S,8R) and that of
P. myxofaciens containing a different uronic acid (
D
-GlcA
vs.
D
-GalA) (Table 3). These data are in full agreement with
the results of serological studies with Pr. rustigianii O14
O-antiserum [28]. In contrast, P. myxofaciens O-antiserum
predominantly contains antibodies to an epitope (or
epitopes) different from AlaLys as it retained the reactivity
with the homologous LPS after absorption with the LPS of
P. mirabilis O13 or Pr. rustigianii O14 (Table 3). The lack
of cross-reactivity of the Pr. alcalifaciens O23 LPS suggests
that epitope(s) associated with AlaLys is feebly exposed on
the LPS of this strain.
Western blot (Fig. 5) also showed that both O-antisera
tested contain antibodies that clearly recognized fast moving
species of the homologous LPS and that these LPS bands of
Pr. rustigianii O14 lack the O-polysaccharide chain and
consist only of core and lipid A moieties. Therefore,
epitopes on the core region, the structures of which remain
unknown in all strains studied, may contribute to the cross-
reactivity of the Pr. rustigianii O14 LPS.
In summary, the structural and serological data show
that P. myxofaciens possesses a unique O-antigen among
Fig. 4. Structures of the O-polysaccharides of Proteus and Providencia
containing N
e
-[(R)-1-carboxyethyl]-

L
-lysine and N
e
-[(S)-1-carboxy-
ethyl]-
L
-lysine (2S,8R-AlaLys and 2S,8S-AlaLys, respectively).
Table 2. EIA data of Proteus and Providencia LPS with rabbit poly-
clonal O-antisera against P. myxofaciens and P. mirabilis O13. Data
for the homologous LPS species are italicized.
LPS
Reactivity in
EIA (reciprocal
titer)
Minimal inhibiting
dose in the homologous
test system in EIA (ng)
P. myxofaciens
O-antiserum
P. myxofaciens
O-antiserum/
P. myxofaciens LPS
P. myxofaciens 1024 000 2
P. mirabilis O13 64 000 800
Pr. rustigianii O14 64 000 800
Pr. alcalifaciens O23 1600
P. mirabilis O13
O-antiserum
P. mirabilis O-antiserum/
P. mirabilis LPS

P. myxofaciens 256 000 125
P. mirabilis O13 512 000 1
Pr. rustigianii O14 256 000 62.5
Pr. alcalifaciens O23 2000
Fig. 3. Structures of amides of
D
-glucuronic and
D
-galacturonic acids
with N
e
-[(R)-1-carboxyethyl]-
L
-lysine (1 and 2, respectively) and
D
-gal-
acturonic acid with N
e
-[(S)-1-carboxyethyl]-
L
-lysine (3).
3186 Z. Sidorczyk et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Proteus strains. We suggest it should be classified into a new
Proteus serogroup, O60, of which this strain is the single
representative.
Acknowledgements
This work was supported by the Science Research Committee (KBN,
Poland; grant 6 P04 A 074 20), the Russian Foundation for Basic
Research (grant 02-04-48767) and INTAS (grant YS 2001-2/1).
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Fig. 5. Western blot of Proteus and Providencia LPS with P. myxo-
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P. myxofaciens,PmO13toP. mirabilis O13, and Pr O14 to Pr. rust-
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blood cells were used as control.
Alkali-treated
LPS used
for absorption
Reactivity (reciprocal titer) with LPS from:
P. myxofaciens
P. mirabilis
O13
Pr. rustigianii
O14
P. myxofaciens O-antiserum
Control 1024 000 64 000 64 000
P. myxofaciens <1000 <1000 <1000
P. mirabilis O13 512 000 <1000 <1000
Pr. rustigianii O14 512 000 <1000 <1000
P. mirabilis O13 O-antiserum
Control 256 000 512 000 256 000
P. myxofaciens <1000 32 000 <1000
P. mirabilis O13 <1000 <1000 <1000
Pr. rustigianii O14 <1000 32 000 <1000

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