Structure of the O-polysaccharide and classification of Proteus mirabilis strain G1 in Proteus serogroup O3 Zygmunt Sidorczyk 1 , Krystyna Zych 1 , Filip V. Toukach 2 , Nikolay P. Arbatsky 2 , Agnieszka Zablotni 1 , Alexander S. Shashkov 2 and Yuriy A. Knirel 2 1 Department of General Microbiology, Institute of Microbiology and Immunology, University of Lodz, Poland; 2 N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation The O -chain polysaccharide of the lipopolysaccharide (LPS) of a previously nonclassi fied strain o f Proteus m irabilis termed G1 was studied by sugar analysis and 1 Hand 13 C NMR spectroscopy, including 2D COSY, TOCSY, rota- ting-frame NOE (ROESY), H-detected 1 H, 13 CHMQC,and heteronuclear multiple-bond correlation (HMBC) experi- ments. The following structure of the polysaccharide was established: where D -GalA6( L -Lys) stands for N a -( D -galacturonoyl)- L -lysine. The structure of the O-polysaccharide of P. mirabilis G1 is similar, but not identical, to that of P. mirabilis S1959 and OXK belonging t o serogroup O3. Immunochemical studies with P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera revealed close L PS-based serological relatedness of P. mirabilis G1 and S1959, and therefore it was suggested to classify P. mirabilis G1 in serogroup O3 as a subgroup. P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera also cross-reacted with LPS of P. mirabilis strains from two other serogroups contain- ing D -GalA6( L -Lys) in the O-polysacch aride or in the core region. Keywords: Proteus mirabilis; O-polysaccharide; lipopoly- saccharide; N a -( D -galacturonoyl)- L -lysine; serogroup. Much has been written about the taxonomy o f Proteus since the original pub lication by Hauser in 1885 who established the genus [1]. Currently, the genus Proteus consists of five named species (P. mirabilis, P. penneri, P. vulgaris, P. myxofaciens and P. hauseri) and three unnamed genomospecies 4, 5 and 6 [2,3]. Proteus rods are widespread in the environment a nd make up part of the normal flora of the human gastrointestinal tract. Proteus ran ks third (after Escherich ia and Klebsiella) as the cause of uncomplicated cystitis, pyelonephritis and prostatitis, particularly, in hos- pital-acquired cases [4]. P. mirabilis accounts for approxi- mately 3% of nosocomial infections in the United States where, together w ith P. penneri, it may play a role in s ome diarrhoeal diseases [5]. Recently, it has been suggested that P. mirabilis may p lay an e thiopathogenic role in rheumatoid arthritis [6]. According to the serological specificity of the O-chain polysaccharides (O-antigens) of the lipopolysaccharides (LPS), strains of P. mirabilis and P. vulgaris have been classified into 6 0 O-serogroups [7,8], i ncluding 49 numbered serogroups (O1 to O49) [7]. Recently, immunochemical studies of LPS enabled establishment of a number of additional serogroups for P. penneri strains [9–11]. The serological heterogeneity of Proteus strains is associated with a high diversity of the O-antigen composition and structure [12,13]. A common structural f eature of most Proteus O-antigens studied so far is the presence of hexuronic acids and their amides with amino acids, which often serve as immunodominant g roups [13]. Here, we report on the structure of a new acidic O-polysaccharide from a nonclassified strain P. mirabilis termed G1, which contains an amide of D -galacturonic acid with L -lysine. Based on chemical an d serological data, we propose to classify th is strain in Proteus serogroup O3. MATERIALS AND METHODS Bacterial strains and growth P. mirabilis strains G1 and D52 were kindly provided by J. Gmeiner (Institute for Microbiology and Genetics, Darmstadt, Germany). Strain G1 was a clinical isolate from urine of a woman with bacteriuria and could be classified in none of 49 O-serogroups in the Kaufman– Perch scheme of Proteus [7]. Biochemical properties of both strains were checked in API 20E test, which showed 99.9% identity with the P. mirabilis species. For other s trains used in this work, P. mirabilis O28 (51/57) was purchased from the Czech National Collection of Type Cultures ( CNCTC, Institute of Epidemiology and Microbiology, Prague, Czech Republic), and P. mirabilis S1959 (O3) and its R14 mutant (T-like form) came from the collection of the Correspondence to Z. Sidorczyk, D epartment of Ge neral Microbio- logy, Institute of Microbiology and Immun o logy, University of Lodz, 90–237, Lodz, Poland. Fax a nd Tel.: + 48 42 635 44 67, E-mail: zsidor@taxus.biol.uni.lodz.pl Abbreviations: EIA, enzyme i mmunosorbent assay; D -GalA(l-Lys), N a -( D -galacturonoyl)- L -lysine; D -GlcA, D -glucuronic acid; HMBC, heteronuclear multiple-bond correlation; LPS, lipopolysaccharide; ROESY, rotating-frame NOE spectroscopy. (Received 15 O ctober 2001, revised 2 January 2002, accepted 11 January 2002) Eur. J. Biochem. 269, 1406–1412 (2002) Ó FEBS 2002 Institute of Microbiology and Immunology, University of Lodz, Poland. Dry bacteria w ere obtained from aerated liquid cultures as described [14]. Isolation and degradation of lipopolysaccharide LPS were obtained by extraction of b acterial mass with a hot phenol/water mixture [15] and purified by treatment with aqueo us 5 0% CC l 3 CO 2 Hat4°C followed by dialysis of the supernatant. Alkali-treated LPS were prepared by saponification of LPS with 0.25 M NaOH ( 56 °C, 2 h) followed by precipitation with ethanol. Acid degradation of P. mirabilis G1 LPS was performed with 0.1 M NaOAc/HOAc buffer pH 4.5 at 100 °Cfor 1.5 h. The O-polysaccharide was isolated by gel-permeation chromatography on a column (3 · 65 cm) of Sephadex G-50 (Pharmacia) using 0.05 M pyridinum-acetate buffer pH 4.5 as eluent; monitoring was p erformed using a Knauer differential refractometer (Germany). Anti-(O-polysaccharide) sera Rabbit polyclonal anti-(O-polysaccharide) sera against P. mirabilis G1 and P. mirabilis S1959 were obtained by intravenous imm unization of rabbits every 5 days with 0.25, 0.5 and 1.0 mL bacterial suspension ( 1.5 · 10 10 c.f.u.ÆmL )1 ), boiled at 100 °C f or 2 h. One week after t he last injection, rabbits were bled. The obtained antisera were stored at )20 °C. For passive immunohemolysis, the antisera w ere inactivated a t 56 °C f or 30 min and absorbed with sheep red blood cells. O ne hemolytic unit of anti-(O-polysaccharide) serum was defined as the antibody dilution yielding 50% lysis of sheep red b lood cells. Agglutination test Agglutination i n tubes was performed w ith a suspension of heat-killed Proteus bacteria incubated (24 h at 50 °C) with diluted P. mirabilis G1 anti-(O-polysaccharide) serum. Passive immunohemolysis, inhibition of passive immunohemolysis and absorption Sheep red blood cells were sensitized with a growing concentration of a lkali-treated LPS fo r 30 min at 37 °C, then washed with NaCl/P i pH 7.2 (15 m M Na 2 HPO 4 ,150m M NaCl) and suspended at a concentration 0 .5% in veronal buffer pH 7.3 (1.8 m M sodium 5,5-diethylbarbiturate, 3.1 m M 5,5-diethylbarbituric acid, 150 m M NaCl, 0.5 m M MgCl 2 ,0.15 m M CaCl 2 ). Anti-(O-polysaccharide) serumwas serially twofold diluted with 50 lL veronal buffered saline and, after adding 50 lL antigen suspension and 25 lL guinea-pig complement diluted ( 1 : 20) with veronal buffer, the plate was incubated a t 3 7 °C f or 1 h. T he last dilution of antiserum g iving 50% hemolysis was established as a titre. A total of 25 lL anti-(O-polysaccharide) serum c ontain- ing 2 or 3 h emolytic units of antibodies was incubated with 25 lL twofold serially diluted inhibitor in microtitrate plates. After incubation (15 min, 37 °C), 50 lLsensitized sheep red blood cells and 25 lL complement were added, the plate was incubated (37 °C for 1 h) and the 50% inhibition value of hemolysis was read. In the absorption test, 1 mL anti-(O-polysaccharide) serum d iluted with N aCl/P i (1 : 50) was treated with 100 lL sheep red blood cells (0.2 mL) sensitized w ith the respective antigen (200 lgLPS)for30mininanicebath.After centrifugation, the level of antibodies was evaluated using passive immunohemolysis test. Enzyme immunosorbent assay (EIA) and inhibition of the reaction in EIA Maxi Sorb microtiter plates (U-bottom form, Nunc, Denmark) were coated with LPS (50 ng per well) diluted with NaCl/P i at 4 °C for 16 h and washed with water. Plates were blocked with 2.5% casein in NaCl/P i (incubation with NaCl/P i /casein for 1 h at 37 °C followed by two washing cycles with NaCl/P i ) and anti-(O-polysaccharide) serum diluted appropriately with NaCl/P i /casein was added. After incubation at 37 °C for 1 h and washing, peroxidase- conjugated goat anti-(rabbit IgG) Ig (Sigma) diluted 1 : 1000 with NaCl/P i /casein was added and incubation was continued for 1 h at 37 °C. After washing in NaCl/P i , the plates were washed twice in substrate buffer (0.1 M sodium citrate pH 4 .5). The substrate solution was freshly prepared as follows: 1 mg azino-di-3-ethyl-benzthiazolin- sulfonic acid (Sigma) was dissolved in 1 mL of substrate buffer with ultrasonication for 3 min and then 25 lL0.1% H 2 O 2 was added. After incubation for 30 min at 37 °C, the reaction was stopped by adding aqueous 2% oxalic acid, and t he plates were read using an Easy Beam Reader (SLT Lab instruments, Finland) at 405 nm. The end titre was taken as the highest dilution of antiserum yielding A 405 >0.2. Inhibitor w as serially twofold diluted with 30 lLNaCl/ P i /casein and mixed in V-shaped microtitrate plates (Med- lab, Poland) with an equal volume of antibodies diluted with the same buffer to give A 405 of 1.0–1.6 without adding the inhibitor. After incubation at 37 °C for 15 min, the mixture was transf erred t o EIA plates coated with LPS, and further steps were performed a s described above. SDS/PAGE and Western blot SDS/PAGE and W estern immunoblots w ere carried out according to Laemmli [16]. Briefly, LPS in sample buffer (4 lL per lane) were separated using 3.5% polyacrylamide stacking gel and 12.5% running gel a nd then transferred to a nitrocellulose membrane. The membrane was blocked with 10% s kimmed milk in dot-blot buffer pH 7.4 (50 m M Tris/HCl and 200 m M NaCl)at20 °C for 1 h and incubated with anti-(O-polysaccharide) serum diluted 1 : 300 with the same buffer for 16 h. The reaction was developed with alkaline phosphatase-conjugated goat anti-(rabbit IgG) Ig (Dianova, Germany) diluted 1 : 500 with blotting buffer supplemented with dried skim milk at 20 °Cfor2h. 5-Bromo-4-chloro-3-indoylphosphate p-toluidine and p-nitroblue tetrazolium chloride (Bio-Rad, Poland) were used as substrate. Sugar analysis The polysaccharide was hydrolysed with 3 M CF 3 CO 2 H (100 °C, 4 h), amino and neutral sugars were identified using Biotronik L C-2000 amino-acid a nd s ugar analysers a s Ó FEBS 2002 O-polysaccharide of Proteus mirabilis G1 (Eur. J. Biochem. 269) 1407 described [17]. The absolute configurations of the mono- saccharides were determined by GLC of the acetylated (S)-2-butyl glycosides [18,19] using a Hewlett-Packard 5890 chromatograph equipped with an Ultra 2 capillary column and a temperature gradient of 160–290 °Cat3°CÆmin )1 . NMR spectroscopy Samples were deuterium-exchanged by freeze-drying two times from D 2 O and examined in D 2 Oat45°Cusing internal acetone as reference (d H 2.225, d c 31.45). 1 Hand 13 C NMR spectra were recorded with a Bruker DRX-500 spectrometer equipped with an SGI INDY computer workstation. 2D NMR experiments were performed using standard Bruker software, and XWINNMR program (Bruker) was used to acquire and process data. A mixing time of 200 and 300 ms was used in TOCSY and ROESY experiments, respectiv ely. RESULTS AND DISCUSSION Structural studies The O-polysaccharide was prepared by mild acid degrada- tion of P. mirabilis G1 LPS followed by gel-permeation chromatography on Sephadex G-50. Sugar analysis of the polysaccharide after a cid hydrolysis revealed glucuronic a cid (GlcA)andgalacturonicacid(GalA)intheratio 1:5. Analysis on an amino-acid analyser showed the presence o f 2-amino-2-deoxygalactose and lysine. The D configuration of GalA and GalN was determined by GLC of the acetylated (S)-2-butyl glycosides and the L configuration of lysine by GLC of the acetylated (S)-2-butyl ester. The D configuration of GlcA was established b y a nalysis of 13 C NMR chemical shift data of the polysaccharide (see below). The 13 C NMR spectrum of the polysacc haride (Fig. 1 ) contained signals for four anomeric carbons at d 100.9– 105.5, one nonsubstituted (d 62.4) and one substituted (d 66.7) C-CH 2 OH groups (C6 of GalN, data of attached- proton test [20]), two carboxyl groups at d 172.3 a nd 174.6 (C6 of GlcA and GalA), two carbons bearing n itrogen at d 52.3 and 53.2 ( C2 of GalN), 14 sugar ring carbons bearing oxygen in t he region d 69.0–81.3, two N-acetyl groups (CH 3 at d 23.6, C O a t d 175.9 and 176.2), and six carbons of lysine at d 23.0, 27.4, 31.9, 40.5, 54.3 a nd 177.9 (Table 1, compare published data [21–23]). Accordingly, the 1 HNMRspec- trum of the polysaccharide (Fig. 2) contained s ignals for four anomeric protons at d 4.51–5.20, two N-acetyl groups at d 2.02 and 2.05, and signals for lysine as shown in Table 1. Therefore, t he polysaccharide has a tetrasaccharide repeating unit containing one residue each of D -GlcA and D -GalA, two residues o f D -GalNAc, and L -lysine. A smaller than expec ted relative con tent o f GlcA i n t he polysaccharide hydrolysate could be accounted for by its retention in oligosaccharides with GalN (see the polysaccharide struc- ture below). The 1 Hand 13 C NMR spec tra o f the polysaccharide w ere assigned using 2D COSY, TOCSY, ROESY, 1 H, 13 C HMQC, and HMQC-TOCSY experiments (Table 1 ). The TOCSY spectrum showed correlations between H1 and H2–H5 for GlcA and GalA and between H1 and H2–H4 for both GalNAc residues (GalNAc I and GalNAc II ). The signals for H5 and H6 of GalNAc I were assigned by H4/H5 correlation in the R OESY spectrum a nd H5/H6 c orrelation in the COSY spectrum. The corresponding 13 CNMR signals were found by 1 H, 13 C correlations in the HMQC spectrum, and three remaining signals were assigned to H5/C5, H6a/C6, and H6b/C6 correlations of GalNAc II . J H,H coupling constant values estimated from the 2D COSY an d T OCSY spectra were typical of sugars with the gluco and galacto configurations in the pyranose form [24]. The GlcA residue was identified by a large J 3,4 coupling constant value of  10 Hz, as compared with J 3,4 £ 3Hz for t he other sugars that have the ga la cto configuration. The Fig. 1. 125-MHz 13 C NMR spectrum of the O-polysaccharide of P. mirabilis G1. 1408 Z. Sidorczyk et al. (Eur. J. Biochem. 269) Ó FEBS 2002 GalNAc residues were distinguished by correlation of protons at carbons bearing nitrogen (H2) to the corresponding carbons (C2) revealed by the 1 H, 13 CHMQC experiment. The signals for the carboxyl groups (C6 of GlcA and GalA and C1 of Lys) were assigned by H5/C6 and H2/C1 correlations, respectively, observed in the HMBC spectrum. The spectrum showed also a correlation between H2 o f Lys and C 6 o f G alA, thus demonstrating th e presence of N a -galacturonoyllysine (GalA6Lys). This con- clusion was confirmed by t ypical 13 C NMR chemical shifts for the free carboxyl group of lysine (d 177.9) and the amidated carboxyl group of GalA (d 172.3) (compare published data [21,23]). Relatively large J 1,2 coupling constant values of 7–8 Hz determined from the 1 H NMR spectrum for the anomeric protons at d 4.51–4.55 showed that GlcA and both GalNAc residues are b-linked. The a-linkage was suggested for a poorly resolved H1 signal of GalA that appeared downfield at d 5.20, and was confirmed by t he 13 C NMR chemical shift data (Table 1, compare to published data [23]). Significant d ownfield displacements of the signals for C3 of GalNAc I ,C4ofGlcA,C4andC6ofGalNAc II to d 81.3, 81.3, 75.9, and 66.7, respectively, as compared with their positions in the corresponding nonsubstituted sugars [25], demonstrated the glycosylation pattern. The 13 CNMR chemical shifts for the GalA residue were close t o t hose for the nonsubstituted monosaccharide [23] and, hence, this residue is terminal. The ROESY s pectrum showed a GalA H1/GalNAc II H4 correlation at d 5.20/4.03, and, hence, GalALys is attached to the disubstituted GalNAc II residue as a m onosaccharide side chain. Correlations of the b-linked sugars in the main chain were d ifficult to i nterpret because o f close positions of the H1 r esonances and m ultiple c oincidences of i ntraresidue H1/H3,H5 and interresidue cross-peaks. The HMBC spec- trum contained a GalNAc I H1/GalNAc II C6 at d 4.55/66.7 and two overlapping cross-peaks at 4.51–4.53/81.3, which could be assigned to GalNAc II H1/GlcA C4 and GlcA H1/ GalNAc I C3 correlations. In addition, GalA C1/GalNAc II H4 and GalNAc II C1/GlcA H4 cross-peaks were present at Table 1. 500-MHz 1 H and 125-MHz 13 C NMR ch emical shifts (d, p.p.m.) of the O-polysaccharide o f P. mirabilis G1. Additional chemical sh ifts for NAc are d H 2.02 and 2.05; d C 23.6 (2 Me), 175.9 a nd 176.2 (both CO). Sugar or amino-acid residue H1 H2 H3a, 3b H4 H5 H6a, 6b C1 C2 C3 C4 C5 C6 fi 3)-b- D -GalpNAc I -(1 fi 4.55 4.02 3.85 4.12 3.70 3.78, 3.78 102.4 52.3 81.3 69.0 76.1 62.4 fi 6)-b- D -GalpNAc II -(1 fi 4.51 3.89 3.80 4.03 3.85 3.89, 4.16 102.8 53.2 71.1 75.9 73.4 66.7 4 › fi 4)-b- D -GlcpA-(1 fi 4.53 3.36 3.56 3.73 3.76 105.5 73.7 74.9 81.3 76.9 174.6 a- D -GalpA-(1 fi 5.20 3.86 4.05 4.30 4.96 100.9 69.5 70.1 71.0 72.6 172.3 L -Lys 4.38 1.79, 1.91 1.43 1.68 3.00 177.9 54.3 31.9 23.0 27.4 40.5 Fig. 2. 500-MHz 1 H NMR spectrum of the O-polysaccharide of P. mirabilis G1. Ó FEBS 2002 O-polysaccharide of Proteus mirabilis G1 (Eur. J. Biochem. 269) 1409 d 100.9/4.03 a nd 102.8/3.73, respectively. The other expected interresidue correlations were either not observed ( for GlcA C1) or difficult to interpret unambiguously (for GalA H1 and GlcA C1). The ROESY and HMBC data were in accordance with the 13 C NMR chemical shift data and were sufficient for determination of the full monosaccharide sequence in the repeating unit. A relatively large effect (> 8 p.p.m.) on C1 of GlcA [25] indicated that GlcA and GalNAc in the b-1 fi 3-linked d isaccharide fragment have t he same absolute configuration (in case of their different absolute configuration the effect on C1 would be about < 5 p.p.m. [26]). Hence, GlcA has the D configuration. On the basis of the data obtained, it was concluded that the O-polysaccharide P. mirabilis G1 has the structure shown in Fig. 3. This structure is similar to that of the O-polysaccharide of P. mirabilis S1959 and OXK from serogroup O3 [22,23], the r epeating unit of P. mirabilis G1 differing on ly in the absence of the lateral a- D -Glcp residue (Fig. 3). Serological studies Rabbit polyclonal anti-(O-polysaccharide) serum against P. mirabilis G1 was tested in immunohemolysis with LPS from the complete s et of Proteus stra ins, including 37 strains of P. mirabilis and 28 strains of P. vulgaris belonging to 49 Proteus O-serogroups as well as 133 strains of P. penneri. From 188 tested LPS, anti-(O-polysaccharide) serum against P. mirabilis G1 reacted only with the homologous LPS and LPS of P. mirabilis S1959, O28, and a mutant of S1959 (R14, T-like form). In enzyme immunosorbent assay (EIA), P. mirabilis G1 and P. mirabilis S1959 anti-(O-polysaccharide) sera showed the strongest reaction w ith LPS of both P. mirabilis G1 and S1959, whereas LPS of P. mirabilis O28 and R14 reacted markedly weaker (Fig. 4). The specificity of the cross- reactions was confirmed b y i nhibition of the reaction in EIA Fig. 3. Structures of the O-polysaccharides of the cross-reactive LPS of P. mirabilis G1, S1959, and O28. Fig. 4. Reactivity of anti-(O-polysaccharide) sera against P. mirabilis G1 (A) and S1959 (B) in EIA. j,LPSofP. mirabilis G1; d,LPSof P. mirabilis S195 9; h,LPSofP. mirabilis O 28; s,LPSofP. mirabilis R14. Antigen do se is 50 ng. Table 2. Reactivity of absorbed anti-(O-polysaccharide) sera against P. mirabilis G1andS1959withalkali-treatedP. mirabilis LPS in E IA . Sheep red blood cells were used as control. Origin of alkali-treated LPS Reciprocal titre for alkali-treated LPS from P. mirabilis S1959 P. mirabilis O28 P. mirabilis R14 P. mirabilis G1 P. mirabilis G1 anti-(O-polysaccharide) serum Control 512 000 256 000 32 000 32 000 P. mirabilis G1 <1000 <1000 <1000 <1000 P. mirabilis S1959 4000 <1000 <1000 <1000 P. mirabilis O28 32 000 32 000 <1000 <1000 P. mirabilis R14 32 000 32 000 <1000 <1000 P. mirabilis S1959 anti-(O-polysaccharide) serum Control 256 000 256 000 64 000 64 000 P. mirabilis G1 2000 <1000 <1000 <1000 P. mirabilis S1959 <1000 <1000 <1000 <1000 P. mirabilis O28 32 000 32 000 <1000 <1000 P. mirabilis R14 32 000 32 000 <1000 <1000 1410 Z. Sidorczyk et al. (Eur. J. Biochem. 269) Ó FEBS 2002 in the homologous systems of P. mirabilis G1 anti-(O- polysaccharide) serum/P. mirabilis G1 LPS and P. mirabilis S1959 anti-(O-polysaccharide) serum/P. mirabilis S1959 LPS. As little as 4–8 ng of the LPS of P. mirabilis G1 and S1959 were sufficient to inhibit the reaction in both test systems, whereas two other cross-reactive LPS were signi- ficantly weaker inhibitors (minimal inhibitory dose 125– 250 ng). The reactivity of P. mirabilis G1 and S1959 anti- (O-polysaccharide) sera in EIA was completely abolished when antisera were absorbed with the homologous LPS (Table 2). Absorption of P. mirabilis G1 anti-(O-polysac- charide) serum with P. mirabilis S1959 LPS significantly decreased the serum titre in the homologous system and completely removed all cross-reactive antibodies against LPS of P. mirabilis S1959, R14 and O28. Absorption with LPS from each of two last strains decreased the reactivity level with L PS of P. mirabilis G1 a nd S 1959 and c ompletely abolished the reactivity with LPS of P. mirabilis R14 and O28. Similar results were obtained with P. mirabilis S1959 anti-(O-polysaccharide) serum absorbed with P. mirabilis G1 LPS (Table 2). These results suggested the presence in P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera of cross-reactive antibodies of at least two types. Antibodies of the first type bound to an epitope on LPS of P. mirabilis G1 and S1959. Antibodies of the other type bound to another epitope shared by the homologous LPS and LPS of all cross- reactive strains. In Western b lot, P. mirabilis G1 anti-(O-polysaccharide) serum recognized slow migrating bands of three LPS (without R14) and fast migrating bands of P. mirabilis G1, O 28, and R14 LPS (Fig. 5A). These bands correspond to high- and low-molecular-mass LPS species consisting of the core-lipid A moiety with or without an O-chain polysaccharide attached, respectively. The lack of reactivity of fast migrating b ands of P. mirabilis S1959 LPS indicated a difference between the core structures in P. mirabilis S1959 and G1. Anti-(O-polysaccharide) serum against P. mirabilis S1959 recognized fast migrating bands of all LPS tested and slow migrating bands of P. mirabilis S1959, G1, and O28 LPS, the banding patterns being clearly different (Fig. 5B). These findings together showed that the epitope shared by P. mirabilis G1 and S1959 is located in the O -chain polysacchar ide, whereas epitopes shared by two other cross-reactive strains are e xposed either on the core or both on the core an d the O-polysaccharide p art of LPS. The serological relatedness of P. mirabilis G1 and S 1959 correlated with the similarity of the chemical structures of their O-polysaccharides (Fig. 3). Therefore, it is reasonable to classify P. mirabilis G1 into the same serogroup O3 as P. mirabilis S1959 [22] and to divide this s erogroup into two subgroups, O 3a,3b for strains P. mirabilis S1959 and OXK [22] and O3a,3c for strain P. mirabilis G1. The major, common epitope O3a is a ssociated with a common structure present in the O-polysaccharides of both strains, which, most likely, includes a lateral N a -( D -galacturonoyl)- L -lysine [a- D -GalpA6( L -Lys)] residue. Previously, this component was found to play an important role in manifesting the immunospecificity of P. mirabilis LPS antigens [21,27,28], including LPS of P. mirabilis S1959 [28]. A partial epitope O3b is evidently linked t o a lateral a- D -Glcp residue which is present in P. mirabilis S1959 but absent from P. mirabilis G1, and a p artial epitope O3c in P. mirabilis G1 may b e an extended epitope that is masked by the a- D -Glcp residue in P. mirabilis S1959. Comparison of the structures of t he O-chain polysaccha- rides a nd core oligosaccharides [21–23,27,29,30] enabled suggestion that D -GalpA6( L -Lys) is responsible for the cross-reactivity of not only P. mirabilis G1 and S1959 but also P. mirabilis O28 and R14. Indeed, LPS of P. mirabilis O28 is characterized by the presence of a- D -GalpA6( L -Lys) in the O-chain polysaccharide [21] (Fig. 3) and b- D -GalpA6( L -Lys) in the core oligosaccharide [29]. No GalA6Lys is present in the polysac charide c hain (T-antigen) of P. mirabilis R14 LPS [27] but in the LPS core region [27]. However, the level of serological c ross-reactivity of LPS of Fig. 5. Western blot of LPS of P. mirabilis G1, S1959, R14, and O 28 with anti-(O-polysaccharide) sera against P. mirabilis G1 (A) and S1959 (B). Ó FEBS 2002 O-polysaccharide of Proteus mirabilis G1 (Eur. J. Biochem. 269) 1411 P. mirabilis O28 and R14 was much lower compared t o t hat of P. mirabilis G1 and S1959 and the structures of their O-polysaccharides are significantly different [21,27]. There- fore, i n spite of the occurrence of t he common LPS epitopes, these strains should be c lassified separately. ACKNOWLEDGEMENTS This work was supported by the Russian F oundation for Basic Research (grant 99-04-48279) and by the University of Lodz (grant 801). REFERENCES 1. Hauser, G. (1885) U ¨ ber Fa ¨ ulnissbacterien und Deren Bezichungen Zur Septica ¨ mie. Ein Beitrag Zur Morphologie de r Spaltpilze. p. 12. Vogel, Leipzig, Germany. 2. O’Hara, C.M., Brenner, F.W., Streigerwalt, A.G., Hill, B.C., Holmes, B., Grimont, P.A.D., Hawkey, P.M., Penner, J.L., Miller, J.M. & Brenner, D.J. (2000) Classification of Proteus vulgaris biogroup 3 w ith r ecognition of Proteus hauserii sp.nov.nom.rev. &unnamedProteus genomospecies 4, 5 and 6. Int. J. Syst. Evol. Microbiol. 50, 1869–1875. 3. O’Hara, C.M., Brenner, F.W. & Miller, J.M. (2000) Classification, identification, and clinical significance of Pr oteus, Providencia,and Morganella. Clin. Microbiol. Rev. 13, 534–546. 4. Stamm, W.E. (1999) Urinary tract infection. In Clinical Infection Diseases: a P ractical Approach (Root, R.K, ed.), pp. 549–656. 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Biochem. 269) Ó FEBS 2002 . Structure of the O-polysaccharide and classification of Proteus mirabilis strain G1 in Proteus serogroup O3 Zygmunt Sidorczyk 1 , Krystyna Zych 1 ,. classify th is strain in Proteus serogroup O3. MATERIALS AND METHODS Bacterial strains and growth P. mirabilis strains G1 and D52 were kindly provided by J.