Báo cáo Y học: Structural studies on the core and the O-polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide

doi:10.1046/j.1432-1033.2002.02875.x

Eur. J. Biochem. 269, 2194–2203 (2002) (cid:1) FEBS 2002

Structural studies on the core and the O-polysaccharide repeating unit of Pseudomonasaeruginosa immunotype 1 lipopolysaccharide

Olga V. Bystrova1, Aleksander S. Shashkov1, Nina A. Kocharova1, Yuriy A Knirel1, Buko Lindner2, Ulrich Za¨ hringer2 and Gerald B. Pier3 1N. 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

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 O-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-

The structure of the lipopolysaccharide (LPS) of Pseudo- monas aeruginosa immunotype 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 O-polysac- charide repeating unit (B) or with a long-chain O-polysac- charide. Therefore, of two core glycoforms, only glycoform 2 accepts the O-polysaccharide.

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 of D-QuiNAc-1-P to undecaprenyl phosphate followed by synthesis of the repeating O-antigen tetrasaccharide.

Keywords: lipopolysaccharide; core oligosaccharide struc- repeating unit; O-polysaccharide; Pseudomonas ture; aeruginosa.

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 the D configuration unless otherwise stated. One or more phosphorylation sites may be occupied by diphosphate groups. In a minority of the

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 (GlcN or 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)

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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 37 (cid:2)C 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 (cid:2)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 0.1 M sodium acetate buffer pH 4.2 and heated for 13 h at 100 (cid:2)C. 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

Pseudomonas aeruginosa is 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 (cid:1)biological(cid:2) 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 serologically distinguishable O-polysaccharides of all 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. aeruginosa 2192 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]:

! 4Þ-a-d-GalpNAcAN3Ac-ð1 ! 4Þ-a-d- GalpNFoAN-ð1 ! 3Þ-a-d-QuipNAc-ð1 ! 2Þ -a-l-Rhap-ð1 !

LPS (200 mg) was treated with anhydrous hydrazine (4 mL) for 1 h at 37 (cid:2)C, then 16 h at 20 (cid:2)C, hydrazine was flushed out in a stream of air at 30–33 (cid:2)C, the residue washed with cold acetone and dried in vacuum. The O-deacylated LPS was dissolved in 4 M NaOH (8 mL), the solution was flushed with nitrogen for 1 h with stirring, heated at 100 (cid:2)C for 16 h, cooled, acidified with concentra- ted HCl to pH 5.5, extracted 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

where GalNAcAN and GalNFoAN stand for 2-acetamido- respectively; and 2-formamido-2-deoxygalacturonamide, 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.

Core oligosaccharide was hydrolyzed with 2 M CF3CO2H (120 (cid:2)C, 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 160 (cid:2)C (3 min) to 290 (cid:2)C at 10 (cid:2)CÆmin)1.

M A T E R I A L S A N D M E T H O D S

Mass spectrometry

Bacterium and cultivation

P. aeruginosa immunotype 1, strain 170041, was from the Hungarian National Collection of Medical Bacteria (National Institute of Hygiene, Budapest, Hungary). It

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.

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Capillary 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 (cid:4) 20 ngÆlL)1 and sprayed with a flow rate of 2 lLÆmin)1.

NMR spectroscopy

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 (cid:4) 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 NMR spectra were obtained on a Bruker DRX-500 spectrometer at 30 (cid:2)C 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, dC 31.45) or external aqueous 85% H3PO4 (dP 0.0). 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 oligosaccharide mixture, both eluting as wide peaks. Sugar analysis of the oligosaccharide product revealed Glc, Rha, and L-glycero-D-manno-heptose (Hep) in the ratios (cid:4) 4 : 2 : 1, respectively, as well as a trace amount of GalN. These monosaccharides are typical components of P. aeruginosa LPS 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 GlaN is accounted for by its N-acylation with L-alanine [5–7].

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)3Hep2Kdo2P5 and that with a DHexNA- QuiN disaccharide 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 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 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 QuiN and two rhamnose residues at d 1.26–1.34, and a proton at the double bond (H4) of DHexNA at d 5.98. Accordingly, the 13C-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 QuiN and 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. The 31P-NMR spectrum of the product contained signals for five monophosphate monoesters at d )1.8, )0.1, 0.2, and 1.3 (two phosphorous). The 1H- and 13C-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 J1,2 coupling constant values and those of Rha, HepI, HepII, KdoI, and KdoII from typical 1H 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

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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.

glucose residues (GlcI–ClcIII) and one residue each of rhamnose and GalN (Fig. 4, Table 1). In contrast, Hep, Kdo and GlcN residues 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]

The remainder of the degraded first repeating unit of the O-polysaccharide was shown to be a b-DHexNA-(1 fi 3)- b-D-QuiN disaccharide attached to position 3 of the terminal Rha residue in glycoform 2 (Fig. 5B). This followed from DHexNA H1/QuiN H3 and QuiN H1/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- Hex3(HexNAla)Hep(HepCm)aKdo] (A), the core oligosaccharide with one O-polysac- charide repeating unit [MI, 6dHexNAc (HexNAcAN)(HexNFoAN)(6dHex)2Hex3 (HexNAla)Hep(HepCm) aKdo] (B), and [M-3H]3– pseudomolecular ions and fragment ions from the reducing end (C), which repre- sent all essentials 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. MP1, MP1Ac1, etc., refer to the molecular ions with one phosphate group and no or one O-acetyl group, YP1, ZP2, etc., refer to the fragment ions with one and two phosphate groups, respectively.

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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.

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).

Fig. 3. Charge deconvoluted negative ion ESI FT-ICR mass spectrum of alkaline degradation products. M and MI belong to the core oligo- saccharide 6dHexHex3(HexN)3Hep2Kdo2P5 and the core oligosaccharide with a remainder of the O-polysaccharide repeating unit DHexNA(6dHexN)6dHexHex3 (HexN)3 Hep2Kdo2P5, respectively.

D I S C U S S I O N

the structure of

well as DHexNA H1/QuiN C3, QuiN H1/Rha C3, DHex- NA C1/QuiN H3, and QuiN C1/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 QuiN residue has the b configuration (dH1 5.06, J1,2 (cid:4) 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].

Previously, 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

The positions of the phosphate groups were determined using a 1H,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.

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Fig. 4. 1H NMR spectrum of alkaline degradation products. Arabic numerals refer to protons in sugar residues. Designations for GlcII, GlcIII, 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

5.74 4.84 2.07 1.85 5.36 5.14 3.47 3.14 2.24 2.09 4.53 4.41 3.92 3.89 4.11 4.13 4.18 4.22 3.63 3.85 4.29 4.08 4.50 3.84 4.11 3.76 3.73 3.73 4.28 4.01 3.60 3.69 3.80 3.73 3.82 3.51 3.89 3.96 3.94 3.80 4.28 3.73 3.85 4.11 3.98 4.54

3.88 3.80 3.78 3.78 5.58 4.64 4.99 4.97 4.79 3.84 3.27 3.51 3.57 4.01 4.45 3.53 3.70 3.68 3.80 4.40 3.28 3.61 3.46 3.44 4.20 3.68 4.17 3.65 3.73 3.88 3.90 3.89 3.85 1.31

3.88 3.90 3.84 3.73 5.58 4.66 5.03 5.00 5.16 3.84 3.49 3.51 3.56 4.25 4.59 3.65 3.76 3.70 4.01 4.40 3.53 3.52 3.40 3.65 4.20 3.68 4.03 3.67 4.03 3.88 3.90 3.84 3.86 1.26

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

approaches. 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.

3.62 1.34 5.06 5.65 3.32 3.73 4.20 4.52 3.51 5.98 Inner core-lipid A backbone fi 6)-a-D-GlcpNI-1-P fi 6)-b-D-GlcpNII-(1 fi fi 4,5)-a-KdopI-(2 fi a-KdopII-(2 fi fi 3)-a-HeppI2,4P-(1 fi fi 3)-a-HeppII6P-(1 fi Outer core, glycoform 1 fi 3,4)-a-D-GalpN-(1 fi fi 6)-b-D-GlcpI-(1 fi fi 6)-a-D-GlcpII-(1 fi a-D-GlcpIII-(1 fi a-L-Rhap-(1 fi Outer core, glycoform 2 fi 3,4)-a-D-GalpN-(1 fi fi 3,6)-b-D-GlcpI-(1 fi a-D-GlcpII-(1 fi a-D-GlcpIII-(1 fi fi 3)-a-L-Rhap-(1 fi Remainder of the O-polysaccharide repeating unit fi 3)-b-D-QuipN-(1 fi b-L-DHexpNA-(1 fi

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Table 2. 13C-NMR data (d, p.p.m.).

Sugar residue C1 C2 C3 C4 C5 C6 C7 C8

64.9 64.3

92.9 100.2 174.2a 174.2a 98.6 103.2 55.2 56.7 100.1 102.4 75.6 70.5 70.3 70.3 64.4 62.7 70.5 72.8 35.3 36.0 75.3 78.9 70.7 75.5 72.2 66.8 74.2 67.7 73.8 75.0 69.5 67.7 73.3 72.6 70.5 63.7 73.1 73.2 71.8 71.8

98.3 105.7 100.6 99.6 102.3 51.8 74.6 73.0 72.4 71.4 77.8 76.9 74.0 74.4 71.6 76.8 71.5 70.1 70.5 73.3 73.7 75.8 71.6 73.5 70.0 61.1 68.8 67.7 61.7 18.3

98.3 105.6 100.5 99.3 101.7 51.8 74.9 73.0 72.4 71.7 77.8 83.2 73.8 74.4 80.7 76.8 69.5 70.3 70.8 72.3 73.7 75.8 72.6 73.5 70.1 61.1 68.8 61.5 62.1 17.6

a Assignment could be interchanged.

101.1 97.4 55.9 53.3 80.1 63.3 76.1 107.3 73.5 147.1 17.7 174.7a Inner core-lipid A backbone fi 6)-a-D-GlcpNI-1-P fi 6)-b-D-GlcpNII-(1 fi fi 4,5)-a-KdopI-(2 fi a-KdopII-(2 fi fi 3)-a-HeppI2,4P-(1 fi fi 3)-a-HeppII6P-(1 fi Outer core, glycoform 1 fi 3,4)-a-D-GalpN-(1 fi fi 6)-b-D-GlcpI-(1 fi fi 6)-a-D-GlcpII-(1 fi a-D-GlcpIII-(1 fi a-L-Rhap-(1 fi Outer core, glycoform 2 fi 3,4)-a-D-GalpN-(1 fi fi 3,6)-b-D-GlcpI-(1 fi a-D-GlcpII-(1 fi a-D-GlcpIII-(1 fi fi 3)-a-L-Rhap-(1 fi Remainder of the O-polysaccharide repeating unit fi 3)-b-D-QuipN-(1 fi b-L-DHexpNA-(1 fi

accordance 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- PP to 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.

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 a1 fi 2-, a1 fi 3-, and b1 fi 3-linkages between QuiNAc and Rha within the O-polysaccharide.

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 QuiNAc to Rha (at position 2 or 3) as well as with

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

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.

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Fig. 6. 2D 1H,31P 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.

(serogroup O6) the terminal 1 fi 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 1 fi 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 1 fi 6-linked Rha blocks the attachment of the 1 fi 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 HepI and 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 HepI at positions 2, 4, and 6, whereas HepII is nonphos- phorylated. Such a pattern seems to be unlikely because the release of 7-O-carbamoylated HepII is 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

and thus plays the same role in serogroup O5 as D-QuiNAc in serogroup O6. Interestingly, WbpL that transfers to Und-P in D-FucNAc-1-P from UDP-D-FucNAc 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

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.

2202 O. V. Bystrova et al.

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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].

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.

which 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.

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.

A C K N O W L E D G E M E N T S

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.

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.).

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