The site and the configuration of the linkage between the polysaccharide and the core and the structure of the O-polysaccharide repeating unit were defined in P.. Keywords: lipopolysacchar
Trang 1Structural studies on the core and the O-polysaccharide repeating
Olga V Bystrova1, Aleksander S Shashkov1, Nina A Kocharova1, Yuriy A Knirel1, Buko Lindner2,
Ulrich Za¨hringer2and Gerald B Pier3
1
N D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia;2Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany;3Channing Laboratory, Department of Medicine,
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
The structure of the lipopolysaccharide (LPS) of
Pseudo-monas aeruginosaimmunotype 1 was studied after mild acid
and strong alkaline degradations by MS and NMR
spec-troscopy Three types of LPS molecules were found,
inclu-ding those with an unsubstituted glycoform 1 core (A) or an
isomeric glycoform 2 core substituted with one
charide repeating unit (B) or with a long-chain
O-polysac-charide Therefore, of two core glycoforms, only glycoform 2
accepts the O-polysaccharide
In the structures A and B, Kdo, Hep, Hep7Cm,
GalNAcAN3Ac, GalNFoAN, QuiNAc, GalNAla
repre-sent 3-deoxy-D-manno-octulosonic acid,L-glycero-D
-manno-heptose, 7-O-carbamoyl-L-glycero-D-manno-heptose,
2-acet-amido-3-O-acetyl-2-deoxygalacturonamide,
2-formamido-2-deoxygalacturonamide, 2-acetamido-2,6-dideoxyglucose
and 2-(L-alanylamino)-2-deoxygalactose, respectively; all
sugars are in the pyranose form and have theDconfiguration
unless otherwise stated One or more phosphorylation sites
may be occupied by diphosphate groups In a minority of the
LPS molecules, an O-acetyl group is present in the outer core region at unknown position
The site and the configuration of the linkage between the polysaccharide and the core and the structure of the O-polysaccharide repeating unit were defined in P aeruginosa immunotype 1 The QuiNAc residue linked to the Rha residue of the core was found to have the b configuration, whereas in the interior repeating units of the
O-polysac-charide this residue is in the a-configuration The data obtained are in accordance with the initiation of biosynthesis
of the O-polysaccharide of P aeruginosa O6, which is closely related to immunotype 1, by transfer ofD-QuiNAc-1-P to undecaprenyl phosphate followed by synthesis of the repeating O-antigen tetrasaccharide
Keywords: lipopolysaccharide; core oligosaccharide struc-ture; repeating unit; O-polysaccharide; Pseudomonas aeruginosa
Correspondence to Y A Knirel, N D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, 119991 Moscow, GSP-1, Russia Fax: + 7095 1355328 E-mail: knirel@ioc.ac.ru
Abbreviations: aKdo, anhydro form of 3-deoxy- D -manno-octulosonic acid; Cm, carbamoyl; FT-ICR, Fourier transform ion cyclotron resonance;
Fo, formyl; Kdo, 3-deoxy- D -manno-oct-2-ulosonic acid; LPS, lipopolysaccharide; OS, oligosaccharide; Hep, L -glycero- D -manno-heptose; HexN, hexosamine (GlcNor GalN); GalNA, 2-amino-2-deoxygalacturonic acid; DHexNA, 2-amino-2-deoxy- L -threo-hex-4-enuronic acid; QuiN, 2-amino-2,6-dideoxy- D -glucose; Und-P, undecaprenyl phosphate.
(Received 28 November 2001, revised 25 February 2002, accepted 11 March 2002)
Trang 2Pseudomonas aeruginosais an opportunistic human
patho-gen, which causes severe infections in hosts with weakened
defense mechanisms often as a result of thermal burns,
surgical operations, or another predisposing disease, such as
cystic fibrosis and cancer [1,2] Lipopolysaccharide (LPS) is
the major surface antigen of P aeruginosa, which plays an
important role in interaction of the bacterium with its host
It is composed of lipid A, a core oligosaccharide, and an
O-chain polysaccharide built up of oligosaccharide
repeat-ing units Lipid A and core are structurally conserved parts
of LPS, whereas the O-polysaccharide is highly variable in
composition and structure The O-specific
heteropolysac-charides are synthesized by assembling individual
mono-saccharides into an oligosaccharide (the so called biological
repeating unit) on an undecaprenyl phosphate (Und-P)
carrier followed by polymerization In several P aeruginosa
strains, the O-polysaccharide is structurally heterogeneous,
most likely, as a result of postpolymerization
nonstoichio-metric modifications, such as O-acetylation, amidation or
epimerization at C5 of uronic acids [3] The structures of the
O-polysaccharides of all serologically distinguishable
smooth (S)-type strains have been determined [3], but the
biological repeating unit was defined only in P aeruginosa
serogroup O5 [4] Structures of the core [5–7] and lipid A
[8,9] of P aeruginosa LPS have also been investigated The
inner core region is composed of two residues of 3-deoxy-D
-manno-oct-2-ulosonic acid (Kdo) and two residues of
L-glycero-D-manno-heptose (Hep), one of which is
specific-ally 7-O-carbamoylated The inner core region is
character-ized by a high degree of phosphorylation but data on the
location of the phosphate groups are contradictory [5–7]
The outer core region contains up to four D-glucose
residues, one L-rhamnose residue, and one residue of
N-(L-alanyl)- or N-acetyl-D-galactosamine; it may include
also O-acetyl groups Recently, it has been reported that
strain P aeruginosa PAO1 and a cystic fibrosis isolate
P aeruginosa2192 produce two different glycoforms of the
LPS outer core [4,5]
Strains belonging to P aeruginosa immunotype 1
(sero-group O6) are frequently isolated from a variety of sources
[3] Previously, the following structure of the
O-polysac-charide of immunotype 1 has been established [3,10–12]:
! GalpNAcAN3Ac-ð1 !
4Þ-a-d-GalpNFoAN-ð1 ! 3Þ-a-d-QuipNAc-ð1 ! 2Þ
-a-l-Rhap-ð1 !
where GalNAcAN and GalNFoAN stand for
2-acetamido-and 2-formamido-2-deoxygalacturonamide, respectively;
QuiNAc stands for 2-acetamido-2,6-dideoxyglucose
In this paper, we present new structural data on the LPS
of P aeruginosa immunotype 1, including elucidation of the
core phosphorylation pattern, the O-polysaccharide
biolo-gical repeating unit, and the site and the mode of the
attachment of the O-polysaccharide to the core
M A T E R I A L S A N D M E T H O D S
Bacterium and cultivation
P aeruginosaimmunotype 1, strain 170041, was from the
Hungarian National Collection of Medical Bacteria
(National Institute of Hygiene, Budapest, Hungary) It
belongs to serogroup O6 of the international antigenic typing system (IATS) and is characterized by an O-antigen factor O6a according to the classification scheme of Lanyi & Bergan [3] Cells were grown in Roux flasks with solid agar medium based on Hottinger broth at 37C for 18 h, then washed in physiological saline, separated by centrifugation, washed with acetone and dried
Isolation of the lipopolysaccharide LPS was isolated from dry bacterial cells by extraction with aqueous 45% phenol (2· 30 min) at 67 C [13] Cells were removed by centrifugation (4000 g, 60 min) The superna-tant was dialyzed against distilled water, nucleic acids were precipitated using Cetavlon [13] and removed by centrifu-gation (5000 g, 90 min) The supernatant was dialyzed against distilled water and lyophilized
Mild acid degradation of the lipopolysaccharide LPS (200 mg) was dissolved in 0.1Msodium acetate buffer
pH 4.2 and heated for 13 h at 100C The precipitate was removed by centrifugation (12 000 g, 10 min), the superna-tant fractionated by gel-permeation chromatography on a column (80· 2.5 cm) of Sephadex G-50 (Pharmacia-Upjohn, Uppsala, Sweden) in pyridinium acetate buffer
pH 4.5 (4 mL pyridine and 10 mL HOAc in 1 L water) at
30 mLÆh)1, monitoring with a Knauer differential refrac-tometer, and 5-mL fraction volume Fractions 24–34 were pooled to give a polysaccharide and fractions 41–54 an oligosaccharide mixture (19.8 and 9.1% of the LPS mass, respectively), the latter containing the core and the core with one O-antigen repeating unit attached
Alkaline degradation of the lipopolysaccharide LPS (200 mg) was treated with anhydrous hydrazine (4 mL) for 1 h at 37C, then 16 h at 20 C, hydrazine was flushed out in a stream of air at 30–33C, the residue washed with cold acetone and dried in vacuum The O-deacylated LPS was dissolved in 4MNaOH (8 mL), the solution was flushed with nitrogen for 1 h with stirring, heated at 100C for 16 h, cooled, acidified with concentra-ted HCl to pH 5.5, extracconcentra-ted twice with dichloromethane, and the aqueous solution desalted by gel chromatography
on Sephadex G-50 The yield of the oligosaccharide fraction was 16.1% of the LPS mass
Composition analysis Core oligosaccharide was hydrolyzed with 2MCF3CO2H (120C, 2 h), monosaccharides were converted into the alditol acetates and analyzed by GLC on a Hewlett-Packard
HP 5890 Series II chromatograph equipped with a 30-m fused-silica SPB-5 column (Supelco), using a temperature gradient of 160C (3 min) to 290 C at 10 CÆmin)1 Mass spectrometry
ESI MS was performed using a Fourier transform ion cyclotron resonance (FT-ICR) mass analyser (ApexII, Bruker Daltonics, USA) equipped with a 7-T actively shielded magnet and an Apollo electrospray ion source
Trang 3Capillary skimmer dissociation was induced by increasing
the capillary exit voltage from )100 to )350 V Samples
were dissolved in a 30 : 30 : 0.01 (v/v/v) mixture of
2-propanol, water, and triethylamine at a concentration of
20 ngÆlL)1and sprayed with a flow rate of 2 lLÆmin)1
NMR spectroscopy
The NMR spectra were obtained on a Bruker DRX-500
spectrometer at 30C in 99.96% D2O Prior to the
measurements, the samples were lyophilized twice from
D2O Chemical shifts are referenced to internal acetone (dH
2.225, dC31.45) or external aqueous 85% H3PO4(dP0.0)
Bruker software XWINNMR 1.2 was used to acquire and
process the data A mixing time of 200 or 100 ms was used
in 2D TOCSY and ROESY experiments, respectively
R E S U L T S
The LPS was delipidated by mild acid hydrolysis at pH 4.2,
and the products were fractionated by gel-permeation
chromatography to give a high-molecular-mass
O-polysac-charide and an oligosacO-polysac-charide mixture, both eluting as
wide peaks Sugar analysis of the oligosaccharide product
revealed Glc, Rha, andL-glycero-D-manno-heptose (Hep) in
the ratios 4 : 2 : 1, respectively, as well as a trace amount
of GalN These monosaccharides are typical components of
P aeruginosaLPS core [5–7]; Rha is also present in the
O-polysaccharide repeating unit [11,12] Most likely, a lower
content of Hep than expected is due to its phosphorylation
and poor release of GlaNis accounted for by its N-acylation
withL-alanine [5–7]
The oligosaccharide was then studied by capillary
skim-mer dissociation ESI FT-ICR MS (Fig 1) The mass
spectrum showed an intense group of [M-H]–
pseudo-molecular ions for core oligosaccharides 6dHexHex3
-(HexNAla)Hep(HepCm)aKdoP0)2Ac0)1 with a Kdo
residue in an anhydro form [14] and a variable number of
phosphate (P0-2) and O-acetyl (Ac0)1) groups (Fig 1A)
The major ion peak at m/z 1590.41 corresponded to the
monophosphorylated non-O-acetylated derivative (the
cal-culated molecular mass 1591.48 Da) A similar series was
observed in the ESI mass spectrum of the core
oligosac-charide from the rough (R)-type LPS of P aeruginosa 2192
[5] In addition, a less intense series of [M-H]–
pseudo-molecular ions was present for the core with one
O-poly-saccharide repeating unit attached (Fig 1B) Again,
heterogeneity of the oligosaccharides was associated with
nonstoichiometric phosphorylation (P0-2) and
O-acetyla-tion (Ac0)2) as well as with incomplete amidation of
GalNAcA or/and GalNAcFo residues resulting in a mass
difference of 1 or 2 Da The major ion peak at m/z
2383.62 corresponded to the monophosphorylated
bisam-idated derivative containing one O-acetyl group (mostly
from the O-polysaccharide repeating unit) having the
calculated molecular mass 2384.75 Da Finally, in the
mass spectrum there was a region of fragment ions from
the reducing end (Y and Z) induced by cleavage of the
linkage between two heptose residues (Fig 1C) They
contained one or two phosphate groups and no O-acetyl
groups Peaks for triple-charged [M-3H]3–
pseudomolecu-lar ions of the core oligosaccharides were in the same
spectral region
The 1H-NMR spectrum of the mild acid degradation oligosaccharide product contained a number of signals for the anomeric protons at d 4.64–5.74, methylene protons of Kdo residues at d 1.9–2.2, methyl groups of 6-deoxy sugars
at d 1.26–1.35 and alanine at d 1.56, as well as N -acetyl and O-acetyl groups Analysis of the spectrum using 2D COSY and TOCSY experiments confirmed the presence of com-ponents of both core and O-polysaccharide repeating unit The position of the major GalNAcAN signals indicated that, like in the O-polysaccharide, this residue is 3-O-acetylated as followed from a downfield displacement of the H3 signal to d 4.89, e.g by 1 p.p.m., due to a deshielding effect of the 3-O-acetyl group An attempt to determine the location of the second, minor O-acetyl group and phosphate groups in the core by NMR spectroscopy failed owing to a high degree of structural heterogeneity
The LPS was O-deacylated by mild hydrazinolysis and N-deacylated by strong alkaline hydrolysis [15] The alkaline degradation was accompanied by depolymerization of the O-polysaccharide by b-elimination in 4-substituted GalNA residues, which were converted into the corresponding hex-4-enuronic acid (DHexNA) (Fig 2) The negative ion mode ESI FT-ICR mass spectrum of the product (Fig 3) showed the presence of the core-lipid A backbone oligosaccharide 6dHexHex3(HexN)3Hep2Kdo2P5and that with a DHexNA-QuiNdisaccharide remainder of the repeating unit of the O-polysaccharide attached (the determined and calculated molecular masses 2357.54 and 2357.51 Da for the former and 2659.64 and 2659.61 Da for the latter compound, respectively) In addition to the major pentakisphosphoryl-ated compounds (P5), there were minor compounds con-taining six (P6) and four (P4) phosphate groups
The 1H-NMR spectrum of the alkaline degradation product (Fig 4, Table 1) contained, among other things, signals for anomeric protons at d 4.64–5.74, axial and equatorial protons (H3) of Kdo residues at d 1.85–2.24, three methyl groups (H6) of one QuiNand two rhamnose residues at d 1.26–1.34, and a proton at the double bond (H4) of DHexNA at d 5.98 Accordingly, the13C-NMR spectrum (Table 2) showed signals for anomeric carbons at
d 92.9–105.7, five nitrogen-bearing carbons (C2 of GlcNI, GlcNII, GalN , QuiN , and DHexNA) at d 51.8–56.7, methyl groups of one QuiNand two rhamnose residues at d 17.6– 18.3, methylene groups (C3) of two Kdo residues at d 35.3 and 36.0, three carboxyl groups (C1 of two Kdo residues and C6 of DHexNA) at d 174.2–174.7, C4 and C5 of DHexNA at d 107.3 and 147.1 The31P-NMR spectrum of the product contained signals for five monophosphate monoesters at d)1.8, )0.1, 0.2, and 1.3 (two phosphorous) The1H- and13C-NMR spectra of the alkaline degrada-tion product were assigned using 2D shift-correlated NMR experiments (COSY, TOCSY, ROESY, and H-detected
1H,13C HMQC) (Tables 1 and 2) The monosaccharide spin systems were assigned based on the coupling constant values and those for amino sugars by correlation of the protons at the nitrogen-bearing carbons to the corresponding carbons The configurations of the glycosidic linkages of the gluco and galacto sugar residues (Glc, GlcN, GalN, QuiN, and DHexNA) followed from the J1,2coupling constant values and those of Rha, HepI, HepII, KdoI, and KdoII from typical1H NMR chemical shifts (compare published data [7,16]) Two series of NMR signals were present for most sugars in the outer core region, which consists of three
Trang 4glucose residues (GlcI–ClcIII) and one residue each of
rhamnose and GalN(Fig 4, Table 1) In contrast, Hep,
Kdo and GlcNresidues in the inner core-lipid A backbone
region gave only one series of signals each These findings
indicated the occurrence of the outer core as two glycoforms
(compare published data [4,5])
Linkage and sequence analyses were performed using 2D
ROESY and HMBC experiments The glycosylation
pat-tern in the core-lipid A backbone region was found to be the
same as in the LPS of P aeruginosa O5 [4,7] and 2192 [5]
with two glycoforms 1 and 2 (Fig 5A and B) As judged by the signal intensities of the methyl groups of Rha and QuiN,
in P aeruginosa immunotype 1 glycoforms 1 and 2 are present in almost equal amounts
The remainder of the degraded first repeating unit of the O-polysaccharide was shown to be a b-DHexNA-(1fi 3)-b-D-QuiNdisaccharide attached to position 3 of the terminal Rha residue in glycoform 2 (Fig 5B) This followed from DHexNA H1/QuiNH3 and QuiNH1/Rha H3 at d 5.65/4.20 and 5.06/4.01 in the ROESY spectrum, as
Fig 1 Negative ion capillary skimmer
disso-ciation ESI FT-ICR mass spectrum of mild acid
degradation products of the LPS Shown are
regions of [M-H] – pseudomolecular ions for
the core oligosaccharide [M,
6dHex-Hex 3 (HexNAla)Hep(HepCm)aKdo] (A),
the core oligosaccharide with one
O-polysac-charide repeating unit [M I , 6dHexN Ac
(HexNAcAN)(HexNFoAN)(6dHex) 2 Hex 3
(HexNAla)Hep(HepCm) aKdo] (B), and
[M-3H] 3– pseudomolecular ions and fragment
ions from the reducing end (C), which
repre-sent all esrepre-sentials ion peaks found in the
complete spectrum, except for peaks for
doubly charged pseudomolecular ions An
explanation of the fragments is shown at the
top of the region C M P1 , M P1Ac1 , etc., refer to
the molecular ions with one phosphate group
and no or one O-acetyl group, Y P1 , Z P2 , etc.,
refer to the fragment ions with one and two
phosphate groups, respectively.
Trang 5well as DHexNA H1/QuiNC3, QuiNH1/Rha C3,
DHex-NA C1/QuiNH3, and QuiNC1/Rha H3 correlations at d
5.65/80.1, 5.06/80.7, 97.4/4.20, and 101.1/4.01 in the HMBC
spectrum, respectively Remarkably, the QuiNresidue has
the b configuration (dH15.06, J1,2 8 Hz), whereas in the
interior repeating units of the O-polysaccharide, this sugar is
in the a-configuration [11,12] The terminal Rha residue in
glycoform 1 is not substituted (Fig 5A) as confirmed by the
C2–C6 chemical shifts (Table 2) being close to those in free
a-rhamnopyranose [17]
The positions of the phosphate groups were determined
using a1H,31P HMQC experiment (Fig 6), which showed
three-bond correlations for the phosphorus signals with
the H1 signals of GlcNI, H4 of GlcNII, H2 and H4 of
HepI, and H6 of HepII at d)1.80/5.74, 0.18/3.85, )0.1/
4.53, 1.30/4.50, and 1.33/4.54, respectively The assignment
of the HepI P2/H2 and HepII P6/H6 cross-peaks was
confirmed by HepI P2/H3 and HepII P6/H5 four-bond
correlations
Therefore, the alkaline degradation products have struc-tures shown in Fig 5 Taking all of the data together, the structures of the core and core with one O-polysaccharide repeating unit in the LPS of P aeruginosa immunotype 1 were established (Fig 7)
D I S C U S S I O N
Previously, the structure of the O-polysaccharide of
P aeruginosa immunotype 1 LPS was elucidated [3,10– 12] However, it cannot be ascertained from this structure what are the first and last monosaccharides in the repeating unit, nor which monosaccharide would be linked to the LPS core The actual biological repeating unit represents the properly ordered oligosaccharide, which, after having been preassembled on an Und-P carrier, is polymerized into the O-polysaccharide In this work, the structure of the biological repeating unit was established by studies of the LPS degradation products prepared using two different
Fig 3 Charge deconvoluted negative ion ESI FT-ICR mass spectrum of alkaline degradation products M and MIbelong to the core oligo-saccharide 6dHexHex 3 (HexN) 3 Hep 2 Kdo 2 P 5 and the core oligosaccharide with a remainder
of the O-polysaccharide repeating unit DHexNA(6dHexN)6dHexHex 3 (HexN) 3 Hep 2 Kdo 2 P 5 , respectively.
Fig 2 Alkaline degradation of the LPS The glycosidic linkage of QuiNAc is a between the O-polysaccharide repeating units and b between the O-polysaccharide and the core.
Trang 6approaches One was mild acid degradation, which,
together with a long-chain polysaccharide, resulted in an
oligosaccharide mixture containing a core oligosaccharide
and that with one O-polysaccharide repeating unit
at-tached The other was strong alkaline degradation, which
caused b-elimination in GalNA residues present in the
O-polysaccharide to give a core oligosaccharide with a
truncated single O-polysaccharide repeating unit from the
LPS species with both long-chain O-polysaccharide and
one repeating unit
Analysis of the products with one O-polysaccharide repeating unit or its remainder by ESI MS and NMR spectroscopy enabled determination of not only the biolo-gical repeating unit but also of the mode and the site of the linkage between the O-polysaccharide and the core It was found that the QuiNAc residue, which occupies the reducing end of the biological repeating unit, has the b con-figuration when linking the O-polysaccharide to the core but the a configuration when connecting the repeating units to each other in the O-polysaccharide This finding is in
Fig 4 1 H NMR spectrum of alkaline degradation products Arabic numerals refer to protons in sugar residues Designations for Glc II , Glc III , and Rha in glycoforms 1 and 2 are not italicized and italicized, respectively.
Table 1.1H-NMR data (d, p.p.m.).
Sugar residue
H1 H3ax
H2 H3eq
H3 H4
H4 H5
H5 H6
H6(6a) H7
H6b(7a) H8a
H7b H8b Inner core-lipid A backbone
fi 6)-a- D -GlcpN I -1-P 5.74 3.47 3.92 3.63 4.11 4.28 3.82
fi 6)-b- D -GlcpN II -(1 fi 4.84 3.14 3.89 3.85 3.76 3.73 3.51
fi 4,5)-a-Kdop I
-(2 fi 2.07 2.24 4.11 4.29 3.73 3.85 3.89 3.60 a-Kdop II -(2 fi 1.85 2.09 4.13 4.08 3.73 4.11 3.96 3.69
fi 3)-a-Hepp I 2,4P-(1 fi 5.36 4.53 4.18 4.50 4.28 3.98 3.94 3.80
fi 3)-a-Hepp II
6P-(1 fi 5.14 4.41 4.22 3.84 4.01 4.54 3.80 3.73 Outer core, glycoform 1
fi 3,4)-a- D -GalpN-(1 fi 5.58 3.84 4.45 4.40 4.20 3.88 3.88
fi 6)-b- D -Glcp I -(1 fi 4.64 3.27 3.53 3.28 3.68 3.90 3.80
fi 6)-a- D -GlcpII-(1 fi 4.99 3.51 3.70 3.61 4.17 3.89 3.78
a- D -Glcp III -(1 fi 4.97 3.57 3.68 3.46 3.65 3.85 3.78
a- L -Rhap-(1 fi 4.79 4.01 3.80 3.44 3.73 1.31
Outer core, glycoform 2
fi 3,4)-a- D -GalpN-(1 fi 5.58 3.84 4.59 4.40 4.20 3.88 3.88
fi 3,6)-b- D -Glcp I -(1 fi 4.66 3.49 3.65 3.53 3.68 3.90 3.90
a- D -GlcpII-(1 fi 5.03 3.51 3.76 3.52 4.03 3.84 3.84
a- D -GlcpIII-(1 fi 5.00 3.56 3.70 3.40 3.67 3.86 3.73
fi 3)-a- L -Rhap-(1 fi 5.16 4.25 4.01 3.65 4.03 1.26
Remainder of the O-polysaccharide repeating unit
fi 3)-b- D -QuipN-(1 fi 5.06 3.32 4.20 3.51 3.62 1.34
b- L -DHexpNA-(1 fi 5.65 3.73 4.52 5.98
Trang 7accordance with the biosynthesis pathway of
O-polysac-charides, which involves multiple enzymes that mediate the
formation of the QuiNAc glycosidic linkage One of them,
glycosyltransferase WbpL, transfers D-QuiNAc-1-P from
UDP-D-QuiNAc to Und-P to initiate the O-polysaccharide
repeating unit biosynthesis in P aeruginosa O6 [18], which is
closely related to P aeruginosa immunotype 1 Another
enzyme, O-antigen polymerase Wzy, mediates
polymeriza-tion of the preassembled oligosaccharide attached to
Und-PPto form a long-chain O-polysaccharide Finally, ligase
WaaL ligates the preassembled oligosaccharide or the
long-chain O-polysaccharide to the core-lipid A moeity
Differences between O-polysaccharide structures of
P aeruginosa immunotype 1 and related serotypes of
P aeruginosa O6 are associated with the configuration
of the QuiNAc linkage (a or b) and the site of attachment of
QuiN Ac to Rha (at position 2 or 3) as well as with
O-acetylation and amidation of the GalNA derivatives (Table 3) Because O-acetylation and amidation, which are nonstoichiometric, are likely to be postpolymerization modifications, it is possible that the oligosaccharide assem-bled on Und-PP is the same in all the serogroup O6 strains and its biosynthesis involves the same WbpL protein and other glycosyltransferases (WbpT, WbpU, and WbpR for
D-GalNAcA,D-GalNFoA, and Rha, respectively [18]) In contrast, the O-antigen polymerase Wzy, or other putative protein(s) that influence the activity of Wzy [18], must be divisible into at least three types in order to adopt the a1fi 2-, a1 fi 3-, and b1 fi 3-linkages between QuiNAc and Rha within the O-polysaccharide
Previously, the structure of the biological repeating unit
of the O-polysaccharide was elucidated in P aeruginosa O5 [4] This O-polysaccharide includes D-FucNAc, which is located at the reducing end of the biological repeating unit
Table 2 13 C-NMR data (d, p.p.m.).
Inner core-lipid A backbone
fi 6)-a- D -GlcpNI-1-P 92.9 55.2 70.5 70.7 73.8 70.5
fi 6)-b- D -GlcpNII-(1 fi 100.2 56.7 72.8 75.5 75.0 63.7
fi 4,5)-a-Kdop I -(2 fi 174.2 a 100.1 35.3 72.2 69.5 73.1 70.3 64.9 a-Kdop II -(2 fi 174.2 a 102.4 36.0 66.8 67.7 73.2 70.3 64.3
fi 3)-a-Hepp I
2,4P-(1 fi 98.6 75.6 75.3 74.2 73.3 71.8 64.4
fi 3)-a-Hepp II 6P-(1 fi 103.2 70.5 78.9 67.7 72.6 71.8 62.7
Outer core, glycoform 1
fi 3,4)-a- D -GalpN-(1 fi 98.3 51.8 77.8 76.8 73.7 61.1
fi 6)-b- D -GlcpI-(1 fi 105.7 74.6 76.9 71.5 75.8 68.8
fi 6)-a- D -Glcp II -(1 fi 100.6 73.0 74.0 70.1 71.6 67.7
a- D -Glcp III -(1 fi 99.6 72.4 74.4 70.5 73.5 61.7
a- L -Rhap-(1 fi 102.3 71.4 71.6 73.3 70.0 18.3
Outer core, glycoform 2
fi 3,4)-a- D -GalpN-(1 fi 98.3 51.8 77.8 76.8 73.7 61.1
fi 3,6)-b- D -GlcpI-(1 fi 105.6 74.9 83.2 69.5 75.8 68.8
a- D -GlcpII-(1 fi 100.5 73.0 73.8 70.3 72.6 61.5
a- D -Glcp III -(1 fi 99.3 72.4 74.4 70.8 73.5 62.1
fi 3)-a- L -Rhap-(1 fi 101.7 71.7 80.7 72.3 70.1 17.6
Remainder of the O-polysaccharide repeating unit
fi 3)-b- D -QuipN-(1 fi 101.1 55.9 80.1 76.1 73.5 17.7
b- L -DHexpNA-(1 fi 97.4 53.3 63.3 107.3 147.1 174.7 a
a
Assignment could be interchanged.
Fig 5 Structures of the major alkaline degra-dation products of the LPS Glycoform 1 core
is unsubstituted (A) and glycoform 2 core substitued with a remainder of the O-poly-saccharide repeating unit (B) All sugars are in the pyranose form and have the D configur-ation unless otherwise stated.
Trang 8and thus plays the same role in serogroup O5 asD-QuiNAc
in serogroup O6 Interestingly, WbpL that transfers
D-FucNAc-1-P from UDP-D-FucNAc to Und-P in
P aeruginosa O5 showed homology to WbpL in
P aeruginosa O6 and both enzymes possess substrate
specificity for UDP-D-FucNAc and UDP-D-QuiNAc [18]
In strains of most other P aeruginosa serogroups the
O-polysaccharide includes D-QuiNAc or/and D-FucNAc
[3]; therefore, the initiation of the O-polysaccharide
biosyn-thesis may proceed in a similar manner in these strains too
The occurrence of two core glycoforms seems to be a
common feature of all P aeruginosa LPS [4,5] As in the
LPS of P aeruginosa O5 [4], in the LPS of immunotype 1
(serogroup O6) the terminal 1fi 3-linked Rha residue of the core oligosaccharide is the site of the attachment of the O-polysaccharide This residue only occurs in the core glycoform 2, whereas the terminal 1fi 6-linked Rha residue in the other, isomeric glycoform 1 cannot accept the O-polysaccharide No unsubstituted core glycoform 2 was detected, nor a core glycoform with two Rha residues
It could be thus suggested that the attachment of the 1fi 6-linked Rha blocks the attachment of the 1fi 3-linked Rha, which is the acceptor of the O-polysaccharide A competition of the corresponding rhamnosyl transferases may provide a mechanism for regulation of the content of long-chain (S-type) and short-chain (R-type) LPS species on the cell surface by an enhanced synthesis of the appropriate glycoform Like the O-polysaccharide chain length, the content of the LPS species containing the core with one O-polysaccharide repeating unit attached (SR-type LPS) is controlled by the O-antigen chain length regulator Wzz [18], which influences the functioning of O-antigen polymerase and ligase by a mechanism that is not clearly understood
As in P aeruginosa strains studied previously [5–7,19,20], the core of the LPS of P aeruginosa immunotype 1 is distinguished by a high degree of phosphorylation Three major phosphorylation sites were determined in the core, two of which are at positions 2 and 4 of HepIand one at position 6 of HepII This finding is in agreement with the phosphorylation pattern in P aeruginosa strains H4 [6] and
2192 [5] but inconsistent with the data reported previously for P aeruginosa O5 and O6 strains [7] According to the latter data, all three phosphorylation sites are located at HepIat positions 2, 4, and 6, whereas HepIIis nonphos-phorylated Such a pattern seems to be unlikely because the release of 7-O-carbamoylated HepIIis significantly increased
by dephosphorylation [21]; these discrepancies are unlikely
to be due to a strain difference because P aeruginosa O6 and immunotype 1 are closely related
Another feature of the LPS core of P aeruginosa immunotype 1 is O-acetylation In P aeruginosa, O-acety-lation has been recently reported in the core of a rough, serogroup O1-derived cystic fibrosis isolate, strain 2192,
Fig 7 Structures of the major glycoform 1 core (A) and glycoform 2
core substitued with one O-polysaccharide repeating unit (B) All sugars
are in the pyranose form and have the D configuration unless otherwise
stated It is not excluded that one or more phosphorylation sites are
occupied by diphosphate groups In the minor products, the outer core
region includes one O-acetyl group at unknown position
O-Acetyla-tation of GalNAcA and amidation of both GalNA derivatives in the
O-polysaccharide repeating unit are incomplete.
Fig 6 2D 1 H, 31 P HMQC spectrum of
alka-line degradation products Three-bond and
four-bond correlations are shown by positive
and negative levels, respectively Other
four-bond correlations were too weak to be
dis-tinguished from noise signals.
Trang 9which produces an R-type LPS [5] The outer core of this
strain has at least four O-acetylation sites, and the major
LPS species is mono-O-acetylated A similar O-acetylation
pattern with up to five O-acetylation sites has been found in
the core of P aeruginosa immunotype 5, O3a,3b,3c, and
O12 (O V Bystrova, A S Shashkov, N A Kocharova,
Y A Knirel, B Lindner, U Za¨hringer & G B Pier,
unpublished data) In immunotype 1, the outer core has at
least one O-acetylation site and the O-acetyl group is present
in the core of a minority of the LPS molecules The position
of the O-acetyl groups in the core of P aeruginosa LPS, as
well as their biological significance, remains to be
deter-mined
A C K N O W L E D G E M E N T S
This work was supported by the Civilian Research and Development
Foundation (CRDF, USA) grant RB1-2042 (to Y A K and
G B P.), the Sonderforschungsbereich (SFB, Germany) 470 (project
B4) (to U Z.), the Deutsche Forschungsgemeinschaft grant LI-448/1-1
(to B L and U Z.), and NIH grants AI22535 and HL58398 (to
G B P.).
R E F E R E N C E S
1 Pollack, M (1985) Pseudomonas aeruginosa In Principles and
Practice of Infectious Diseases, Part III, Infectious Diseases and
Their Etiologic Agents (Mandell, G.L., Douglas, R.G & Bennet,
J.E., eds), pp 1236–1250 John Wiley, New York.
2 Pier, G.B (2000) Peptides, Pseudomonas aeruginosa,
polysac-charides and lipopolysacpolysac-charides – players in the predicament of
cystic fibrosis patients Trends Microbiol 8, 247–250.
3 Knirel, Y.A (1990) Polysaccharide antigens of Pseudomonas
aeruginosa CRC Crit Rev Microbiol 17, 273–304.
4 Sadovskaya, I., Brisson, J.-R., Thibault, P., Richards, J.C., Lam,
J.S & Altman, E (2000) Structural characterization of the outer
core and the O-chain linkage region of lipopolysaccharide from
Pseudomonas aeruginosa serotype O5 Eur J Biochem 267, 1640– 1650.
5 Knirel, Y.A., Bystrova, O.V., Shashkov, A.S., Kocharova, N.A., Senchenkova, S.N., Moll, H., Lindner, B., Za¨hringer, U., Hatano,
K & Pier, G.B (2001) Structural analysis of the lipopoly-saccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa Eur J Biochem 268, 4708–4719.
6 Sa´nchez-Carballo, P.M., Rietschel, E.T., Kosma, P & Za¨hringer,
U (1999) Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4 Eur J Biochem 261, 500– 508.
7 Sadovskaya, I., Brisson, J.-R., Lam, J.S., Richards, J.C & Altman, E (1998) Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chain-deficient mutant strains AK1401 and AK1012 from Pseudomonas aerugi-nosa serotype O5 Eur J Biochem 255, 673–684.
8 Bhat, U.R., Marx, A., Galanos, C & Conrad, R.S (1990) Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4-deoxyarabinose J Bacteriol 172, 6631– 6636.
9 Kulshin, V.A., Za¨hringer, U., Lindner, B., Ja¨ger, K.E., Dmitriev, B.A & Rietschel, E.T (1991) Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides Eur J Biochem 198, 697–704.
10 Vinogradov, E.V., Knirel, Y.A., Shashkov, A.S & Kochetkov, N.K (1987) Determination of the degree of amidation of 2-deoxy-2-formamido- D -galacturonic acid in O-specific polysaccharides of Pseudomonas aeruginosa O4 and related strains Carbohydr Res.
170, C1–C4.
11 Knirel, Y.A., Shashkov, A.S., Dmitriev, B.A & Kochetkov, N.K (1984) Structural studies of the Pseudomonas aeruginosa immunotype 1 antigen, containing the new sugar constituents: 2-acetamido-2-deoxy- D -galacturonamide and 2-deoxy-2-for-mamido- D -galacturonic acid Carbohydr Res 133, C12–C14.
12 Knirel, Y.A., Vinogradov, E.V., Shashkov, A.S., Dmitriev, B.A., Kochetkov, N.K., Stanislavsky, E.S & Mashilova, G.M (1985) Somatic antigens of Pseudomonas aeruginosa The structure of the Table 3 Structures of the O-polysaccharides of P aeruginosa immunotype 1 and serogroup O6 (Lanyi-Bergan classification) In serotypes O6a,6b and O6a,6d, GalNFoA is amidated to a minor extent (10–20%) [3,10–12].
Trang 10O-specific polysaccharide chains of lipopolysaccharide of
P aeruginosa serogroup O4 (Lanyi) and related serotype O6
(Habs) and immunotype 1 (Fisher) Eur J Biochem 150, 541–550.
13 Westphal, O & Jann, K (1965) Bacterial lipopolysaccharides.
Extraction with phenol-water and further applications of the
procedure Methods Carbohydr Chem 5, 83–91.
14 Olsthoorn, M.M.A., Haverkamp, J & Thomas-Oates, J.E (1999)
Mass spectrometric analysis of Klebsiella pneumoniae ssp
pneu-moniae rough strain R20 (O1–: K20–) lipopolysaccharide
prepa-rations: Identification of novel core oligosaccharide components
and three 3-deoxy- D -manno-oct-2-ulopyranosonic artifacts.
J Mass Spectrom 34, 622–636.
15 Holst, O (2000) Deacylation of lipopolysaccharides and isolation
of oligosaccharide phosphates In Bacterial Toxins Methods and
Protocols (Holst, O., ed.), pp 345–353 Humana Press, Totowa,
New Jersey.
16 Knirel, Y.A., Grosskurth, H., Helbig, J.H & Za¨hringer, U (1995)
Structures of decasaccharide and tridecasaccharide
tetraphos-phates isolated by strong alkaline degradation of O-deacylated
lipopolysaccharide of Pseudomonas fluorescens strain ATCC
49271 Carbohydr Res 279, 215–226.
17 Bock, K & Pedersen, C (1983) Carbon-13 nuclear magnetic resonance spectroscopy of monosaccharides Adv Carbohydr Chem Biochem 41, 27–66.
18 Rocchetta, H.L., Burrows, L.L & Lam, J.S (1999) Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa Microbiol Mol Biol Rev 63, 523–553.
19 Masoud, H., Altman, E., Richards, J.C & Lam, J.S (1994) General strategy for structural analysis of the oligosaccharide region of lipooligosaccharides Structure of the oligosaccharide component of Pseudomonas aeruginosa IATS serotype O6 mutant R5 rough-type lipopolysaccharide Biochemistry 33, 10568–10578.
20 Masoud, H., Sadovskaya, I., De Kievit, T., Altman, E., Richards, J.C & Lam, J.S (1995) Structural elucidation of the lipopoly-saccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype 06 (International Anti-genic Typing Scheme) J Bacteriol 177, 6718–6726.
21 Beckmann, F., Moll, H., Ja¨ger, K.-E & Za¨hringer, U (1995) 7-O-Carbamoyl- L -glycero- D -manno-heptose: a new core constituent
in the lipopolysaccharide of Pseudomonas aeruginosa Carbohydr Res 267, C3–C7.