Eur. J. Biochem. 267, 601±605 (2000) q FEBS 2000
Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 63 from a new serogroup O68 Aleksander S. Shashkov1, Anna N. Kondakova1, Sof'ya N. Senchenkova1, Krystyna Zych2, Filip V. Toukach1, Yuriy A. Knirel1 and Zygmunt Sidorczyk2 1
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia; 2Institute of Microbiology and Immunology, University of Lodz, Poland
Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass Ospecific polysaccharide that was isolated by gel-permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two-dimensional 1H,1H COSY, TOCSY rotating-frame NOE spectroscopy, H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence (HMQC), and 1H,13C HMQC-TOCSY experiments, demonstrated the following structure of the polysaccharide:
where FucNAc is 2-acetamido-2,6-dideoxygalactose and PEtn is 2-aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of d-GlcNAc6PEtn and an a-l-FucNAc-(1!3)-dGlcNAc disaccharide, with other Proteus O-specific polysaccharides. A marked cross-reactivity of P. penneri 63 O-antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O-specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O-antigens, and therefore a new, separate serogroup, O68, is proposed for this strain. Keywords: 2-aminoethyl phosphate; lipopolysaccharide; O-antigen; O-serogroup; Proteus penneri.
Medically important bacteria of the genus Proteus, a common cause of urinary tract infections, are divided into three species, P. mirabilis, P. vulgaris and P. penneri (formerly P. vulgaris biogroup I). Strains of Proteus are serologically heterogeneous due to a high diversity in the composition and structure of the O-specific polysaccharide chain (O-antigen) of the outermembrane lipopolysaccharide (LPS). Based on the specificity of the O-antigens, strains of P. mirabilis and P. vulgaris have been classified into 60 O-serogroups [1,2], and, recently, a number of additional Proteus O-serogroups have been proposed for P. penneri strains [3]. To create a chemical basis for serological classification, we have determined structures of the O-specific polysaccharides of a number of serologically different P. penneri strains [3]. Like O-antigens of P. mirabilis and P. vulgaris, most O-specific polysaccharides of P. penneri contain acidic or both acidic and basic functions [3]. We now report the structure of a new charged O-specific polysaccharide from P. penneri strain 63 and propose for this strain a new Proteus O-serogroup. Correspondence to Y. A. Knirel, N. D. Zelinsky Institute of Organic Chemistry, Leninsky Prospekt 47, Moscow 117913, Russia. Fax: +7095 1355328. E-mail:
[email protected] Abbreviations: FucNAc, 2-acetamido-2,6-dideoxygalactose; HMQC, heteronuclear multiple-quantum coherence; LPS, lipopolysaccharide; PEtn, 2-aminoethyl phosphate; ROESY, rotating-frame NOE spectroscopy. (Received 18 October 1999, revised 22 November 1999, accepted 24 November 1999)
M AT E R I A L S A N D M E T H O D S Bacterial strains and growth P. penneri strain 63 was isolated in Lodz (Poland) from urine of a man with bacteriuria. Other P. penneri strains were from the collection of the Department of General Microbiology, University of Lodz, Poland. Strains of P. mirabilis and P. vulgaris were from the Czech National Collection of Type Cultures (Institute of Epidemiology and Microbiology, Prague). Dry bacterial cells of P. penneri 63 were obtained from an aerated liquid culture as described [4]. Isolation and degradation of LPS A crude LPS preparation was obtained by extraction of the bacterial mass with a hot phenol/water mixture [5] and purified by treatment with cold aqueous 50% CCl3CO2H [6]. Degradation of LPS was performed with aqueous 1% HOAc at 100 8C for 2 h, and products were fractionated by gel-permeation chromatography on Sephadex G-50 (Pharmacia) using 0.05 m pyridine acetate pH 4.5 as eluent; monitoring was performed using a Knauer differential refractometer. Sugar analysis The polysaccharide was hydrolysed with 2 m CF3COOH (120 8C, 3 h). Amino sugars were identified using a Biotronik
602 A. S. Shashkov et al. (Eur. J. Biochem. 267)
LC-2000 amino-acid analyser equipped with a column (0.4 22 cm) of Ostion LG AN B cation-exchange resin as described [7]. Neutral sugars were identified using a Biotronik LC-2000 sugar analyser on a column (0.4 15 cm) of Dionex AX8 anion-exchange resin using 0.5 m sodium borate pH 8.0 at 65 8C. Alditol acetates were derived from the polysaccharide hydrolysate as described [8] and analysed by GLC using a Hewlett-Packard 5890 chromatograph equipped with a DB-5 fused-silica capillary column. The absolute configurations of the monosaccharides were determined by GLC of acetylated (+)-2-octyl glycosides according to the published method [9] modified as described [10]. Methylation analysis The polysaccharide was methylated according to the Hakomori procedure [11]; methylated polysaccharide was recovered by extraction with ethyl acetate, hydrolysed as in sugar analysis; partially methylated sugars were conventionally converted into alditol acetates and analysed by GLC-MS using a NERMAG R10-10L mass spectrometer. Partially methylated alditol acetates were identified using published data [12,13]. In another experiment, prior to methylation the polysaccharide was dephosphorylated using aqueous 48% HF (20 8C, 24 h). NMR spectroscopy 1 H-, 13C- and 31P-NMR spectra were recorded with a Bruker DRX-500 spectrometer in D2O at 60 8C using internal acetone (dH 2.225, dC 31.45) or external aqueous H3PO4 (dC 0) as references. Standard Bruker software (xwinnmr 1.2) was used to acquire and process NMR data. A mixing time of 150 ms was used in TOCSY and heteronuclear multiple-quantum coherence (HMQC)-TOCSY experiments, and 300 ms in a rotating-frame NOE spectroscopy (ROESY) experiment.
O-antiserum and serological techniques Rabbit polyclonal O-antiserum was obtained according to published procedure [14]. Enzyme immunoabsorbent assay using an LPS/BSA complex as solid phase antigen [15], passive immunohemolysis with alkali-treated LPS, absorption, SDS/PAGE and Western blot analysis [16] were performed as described earlier.
R E S U LT S A N D D I S C U S S I O N Chemical and NMR spectroscopic studies of the O-specific polysaccharide O-specific polysaccharide was prepared by mild acid degradation of P. penneri 63 LPS followed by gel-permeation chromatography on Sephadex G-50. Sugar analyses of the polysaccharide revealed glucose, which was identified using a sugar analyser, and three amino components, 2-amino-2-deoxyglucose, 2-amino-2,6-dideoxygalactose (FucN) and 2-aminoethanol in the ratios < 2 : 2 : 1. The identification of the monosaccharides was additionally confirmed by GLC of alditol acetates and acetylated (+)-2-octyl glycosides. Analysis of the latter derivatives showed the l configuration of FucN and the d configuration of the other monosaccharides. The 13C-NMR spectrum of the polysaccharide (Fig. 1) demonstrated a pentasaccharide repeating unit. The spectrum contained signals for five anomeric carbons at d 98.6±104.2, two unsubstituted HOCH2-C groups at d 61.6 and 61.9, two
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substituted HOCH2-C groups at d 66.0 and 69.4 (C6 of Glc and GlcN, data of attached-proton test [17]), one CH3-C group at d 16.7 (C6 of FucN), three carbons bearing nitrogen at d 50.4, 54.5 and 57.0 (C2 of GlcN and FucN), 17 sugar ring carbons bearing oxygen in the region d 68.9±81.2, three N-acetyl groups (CH3 at d 23.4±23.5, CO at d 174.8±175.3), and one residue of 2-aminoethanol (CH3N at d 41.3 and CH2O at d 63.2). Accordingly, the 1H-NMR spectrum of the polysaccharide contained signals for one CH2-C group at d 1.31 (d, J5,6 6 Hz, H6 of FucN), three N-acetyl groups at d 1.96±2.03 (all s), and one residue of 2-aminoethanol (CH2N at d 3.32, t, J 6 Hz). In a low-field region of the spectrum, there were seven signals, five of which at d 4.55±5.05 belonged to anomeric protons (see below). The 31P-NMR spectrum of the polysaccharide contained one signal for a phosphomonoester at d 3.2. Therefore, the polysaccharide has a pentasaccharide repeat unit containing two residues each of d-Glc and d-GlcNAc, one residue of l-FucNAc and one 2-aminoethyl phosphate group (PEtn). Methylation analysis using GLC-MS of partially methylated alditol acetates derived from the dephosphorylated polysaccharide demonstrated terminal and 2-substituted Glc residues, 3- and 6-substituted GlcNAc residues and a 3,4-disubstituted residue FucNAc. The last sugar was identified by a characteristic peak of the C2-C6 fragment ion at m/z 316 present in the mass spectrum of the corresponding derivative. Therefore, the polysaccharide is branched, one of the Glc residues is the lateral sugar, and FucNAc is at the branch point. The 1H-NMR spectrum of the polysaccharide was assigned using two-dimensional NMR. The COSY spectrum displayed clearly H1/H2 and H2/H3 cross-peaks for all five sugar residues. The spin system of FucNAc was identified by a low-field position of the signal for H2 at d 4.46 and its correlation to the signal for C2 at d 50.4, as revealed by a H-detected 1 H,13 C HMQC experiment. The signals for H5 and H4 of FucNAc were assigned by H5/H6 and H4/H5 crosspeaks at d 4.47/1.31 and 4.08/4.47 in COSY and ROESY spectra, respectively. The spin systems of the GlcNAc residues (GlcNAcI and GlcNAcII) were distinguished from those of the Glc residues (GlcI and GlcII) by correlation of protons at carbons bearing nitrogen at d 3.98 and 3.91 to the corresponding carbons at d 54.5 and 57.0 in the 1H,13C HMQC spectrum. The TOCSY spectrum contained cross-peaks of H1 with H2-H6 for all five sugar residues. A 1 H,13 C HMQCTOCSY experiment demonstrated correlations of C1 with H2-H6 of GlcI, GlcII and GlcNAcII, and H2-H5 of GlcNAcI and FucNAc. These data allowed the unambiguous assignment of the 1H-NMR spectrum (Table 1) and the following assignment of the 13C-NMR spectrum of the polysaccharide (Table 2) using 1H,13C HMQC (Fig. 2) and HMQC-TOCSY experiments. As judged by a relatively large J1,2 coupling constant values of 7±8 Hz, GlcII and GlcNAcII are b-linked. The coupling constants for the three remaining signals of the anomeric protons were not resolved in the 1H-NMR spectrum because of line broadening or/and higher order effects. Based on low-field positions at d 5.05 and 5.00, two narrower signals of GlcNAcI and FucNAc residues were assigned to H1 of a-linked sugars. A higher-field, broader H1 signal at d 4.55 was assigned to the b-linked GlcI residue. This signal was not resolved because of the coincidence of the signals for H2 and H3 at d 3.55 (Table 1). The configurations of the glycosidic linkages were confirmed by two-dimensional ROESY, which revealed correlations of H1 with H3 and H5 of the b-linked, but not a-linked, sugar residues.
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Fig. 1. 125-MHz
O-specific polysaccharide of Proteus penneri 63 (Eur. J. Biochem. 267) 603
13
C-NMR spectrum of the O-specific polysaccharide.
A H-detected 1H,31P HMQC experiment on the polysaccharide revealed correlation of the phosphorus at d 3.2 with protons of the 2-aminoethanol residue at d 3.32 (CH2N) and 4.17 (CH2O), as well as with H6a,b of GlcNAcII at d 4.13 and 4.25, respectively. These data indicated that the PEtn group is attached at position 6 of GlcNAcII, as confirmed also by a low-field position of the signal for C6 of GlcNAcII at d 66.0. Low-field displacements in the 13C-NMR spectrum of the signals for C3 and C4 of FucNAc, C3 of GlcNAcII, C6 of GlcNAcI and C2 of GlcI to d 71.4, 79.1, 78.9, 69.4 and 81.2, respectively, as compared with their positions in the spectra of the corresponding unsubstituted monosaccharides [18], revealed the modes of monosaccharide glycosylation, which were in agreement with the methylation analysis data (see above). In accordance with the terminal position of GlcII in the side chain, the chemical shifts for C2-C6 of this residue were close to those in unsubstituted b-Glcp [18]. In addition to intra-residue NOE correlations, the ROESY spectrum of the polysaccharide showed a number
of inter-residue correlations. The following correlations between the transglycosidic protons were identified: GlcI H1/GlcNAcI H6 at d 4.55/4.06; GlcNAcI H1/FucNAc H3 at d 5.05/4.10; FucNAc H1/GlcNAcII H3 at d 5.00/3.77; GlcNAcII H1/GlcI H2 at d 4.91/3.55; and GlcII H1/FucNAc H4 at d 4.63/4.08. These data were in agreement with the linkage analysis data and allowed determination of the full sequence of the monosaccharide residues in the repeating unit. Therefore, the O-specific polysaccharide of P. penneri 63 has the structure shown in Fig. 3. The polysaccharide studied has structural features in common with some other Proteus O-specific polysaccharides. For instance, b-d-GlcNAc6PEtn has been found in the O-antigens of P. mirabilis O27 [19], P. penneri O67 (strain 8) [3] and P. penneri strain 31 (A. S. Shashkov, A. N. Kondakova, S. N. Senchenkova, K. Zych, F. V. Toukach, Y. A. Knirel and Z. Sidorczyk, unpublished data); furthermore, an a-l-FucNAc(1!3)-d-GlcNAc disaccharide fragment is present in all Proteus polysaccharides containing FucNAc [3].
Table 1. 500-MHz 1H-NMR chemical shifts for the O-specific polysaccharide (d, p.p.m.). Chemical shifts for the N-acetyl groups are d 1.96, 1.98 and 2.03.
Table 2. 125-MHz 13C-NMR chemical shifts for the O-specific polysaccharide (d, p.p.m.). Chemical shifts for N-acetyl groups are d 23.4, 23.4, 23.5 (Me), 174.8, 175.0 and 175.3 (CO).
Sugar residue
H1
H2
H3
H4
H5
H6a
Sugar residue
!6)-a-d-GlcpNAcI-(1! !3)-a-l-FucpNAc-(1! 4 "
5.05 5.00
3.98 4.46
3.73 4.10
3.72 4.08
3.95 4.47
4.06 1.31
!3)-b-d-GlcpNAcII-(1! !2)-b-d-GlcpI-(1! b-d-GlcpII-(1! Etn
4.91 4.55 4.63 4.17
3.91 3.55 3.45 3.32
3.77 3.55 3.53
3.61 3.42 3.45
3.62 3.44 3.42
4.13 3.73 3.76
H6b
4.25 3.91 3.97
C1
C2
C3
C4
C5
C6
!6)-a-D-GlcpNAcI-(1! !3)-a-L-FucpNAc-(1! 4 "
99.0 98.6
54.5 50.4
72.4 71.4
70.5 79.1
72.2 68.9
69.4 16.7
!3)-b-D-GlcpNAcII-(1!
102.2
57.0
78.9
69.5
75.9
66.0
103.0 104.2 63.2
81.2 75.0 41.3
77.4 77.5
70.8 70.9
76.8 76.8
61.6 61.9
I
!2)-b-D-Glcp -(1! b-D-GlcpII-(1! Etn
604 A. S. Shashkov et al. (Eur. J. Biochem. 267)
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Fig. 2. Part of an 1H,13C HMQC spectrum of the O-specific polysaccharide. The corresponding parts of the 1H-and 13C-NMR spectra are displayed along the horizontal and vertical axes, respectively. Designations refer to the signals for PEtn.
Serological studies with Proteus LPS Rabbit polyclonal O-antiserum against P. penneri 63 was tested with LPS from the complete set of Proteus strains, namely, 37 strains of P. mirabilis and 28 strains of P. vulgaris, which represent 49 Proteus O-serogroups, as well as 68 strains of P. penneri. From the 133 LPS tested, only the homologous LPS and one heterologous LPS, namely, that of P. vulgaris O12, reacted significantly. However, the reactivity of the heterologous LPS was much weaker as compared with the homologous LPS (titres 1 : 6400 and 1 : 51 200 in passive immunohaemolysis; 1 : 32 000 and 1 : 512 000 in enzyme immunobsorbent assay, respectively). Absorption of the P. penneri 63 O-antiserum with alkalitreated LPS of P. penneri 63 removed completely the homologous and cross-reacting antibodies, whereas absorption with alkali-treated LPS of P. vulgaris O12 merely decreased the reactivity with the homologous LPS in passive immunohaemolysis from the titre of 1 : 51 200 to 1 : 12 800. In Western blot after SDS/PAGE separation of the two crossreactive LPS, P. penneri 63 O-antiserum reacted with slowly migrating long-chain polysaccharide-containing LPS species
from both strains. Similarly, P. vulgaris O12 O-antiserum cross-reacted with slowly migrating bands of P. penneri 63 LPS [20]. These data suggest that the cross-reactive LPS have a common epitope (or epitopes) on the polysaccharide moiety. Comparison of the structure of the O-specific polysaccharide of P. penneri 63 established in this work with that of P. vulgaris O12 reported elsewhere [20] (Fig. 3) suggested that, most likely, the common epitope is associated with a b-d-Glcp(1!4)-a-l-FucpNAc-(1!3)-b-d-GlcpNAc trisaccharide fragment shared by the two cross-reactive O-antigens. Structural similarity of the O-specific polysaccharide and the corresponding cross-reactivity of LPS are not uncommon for Proteus strains belonging to different O-serogroups [3]. Based on the finding that the structure of the O-specific polysaccharide of P. penneri 63 is unique among the known structures of Proteus O-antigens and that the cross-reactivity with P. vulgaris O12 is relatively weak, we propose for P. penneri 63 a new, separate Proteus serogroup O68, in which at present this strain is the single representative.
ACKNOWLEDGEMENTS This work was supported by grant 99-04-48279 of the Russian Foundation for Basic Research and grant 6 PO4A 007 14 of the Science Research Committee (KBN, Poland).
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Fig. 3. Structures of the O-specific polysaccharides of the cross-reactive Proteus LPS. In P. vulgaris O12, the degree of O-acetylation at position 4 of GalNAc is < 25%.
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