Báo cáo khoa học: Structures of the O-polysaccharides and classification of Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups

Structures of the O-polysaccharides and classification of Proteus genomospecies 4, 5 and 6 into respective Proteus serogroups Krystyna Zych1, Andrei V. Perepelov2, Małgorzata Siwinˇ ska1, Yuriy A. Knirel2 and Zygmunt Sidorczyk1

1 Department of General Microbiology, Institute of Microbiology and Immunology, University of Ło´ dz´, Poland 2 N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation

Keywords Proteus; O-antigen; O-serogroup; serological classification; 3-acetamido-3,6-dideoxy- D-glucose

An acidic branched O-polysaccharide was isolated by mild acid degrada- tion of the lipopolysaccharide (LPS) of Proteus genomospecies 4 and stud- ied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H-detected 1H,13C HSQC experiments. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established, which is unique among Proteus polysaccharide structures:

Correspondence Z. Sidorczyk, Department of General Microbiology, Institute of Microbiology and Immunology, University of Ło´ dz´, Banacha 12 ⁄ 16, 90–237 Lo´ dz, Poland Tel: +48 42 6354467 Fax: +48 42 6655818 E-mail: zsidor@biol.uni.lodz.pl

(Received 16 May 2005, revised 30 August 2005, accepted 5 September 2005)

doi:10.1111/j.1742-4658.2005.04958.x

where Qui3NAc stands for 3-acetamido-3,6-dideoxyglucose. Based on the O-polysaccharide structure and serological data, we propose classifying Proteus genomospecies 4 into a new, separate Proteus serogroup, O56. A weak cross-reactivity of Proteus genomospecies 4 antiserum with LPS of Providencia stuartii O18 and Proteus vulgaris OX2 was observed and is dis- cussed in view of a similarity of the O-polysaccharide structures. Structural and serological investigations showed that Proteus genomospecies 5 and 6 should be classified into the existing Proteus serogroups O8 and O69, respectively.

Gram-negative bacteria of the genus Proteus are com- mon in human and animal intestines but under favour- able conditions they cause infections of wounds, burns, skin, eyes, ears, nose and throat, as well as intestinal and urinary tract infections. The last illness can lead to severe complications, such as acute or chronic pyelone- phritis and formation of bladder and kidney stones. In the genus Proteus there are four clinically important named species: P. mirabilis, P. vulgaris, P. penneri and P. hauseri as well as three unnamed Proteus genomo- species 4, 5 and 6 [1].

The outer membrane lipopolysaccharide (LPS) is a somatic antigen and is considered to be one of the most important potential virulence factors of Proteus [2]. The serological O-specificity of Gram-negative bacteria is defined by the structure of the polysaccha- ride chain of LPS (O-antigen). Based on the somatic antigens, two Proteus species: P. mirabilis and P. vul- garis, have been classified into 49 O-serogroups [3]. This classification, however, does not include two groups of P. mirabilis and P. vulgaris representatives (totally 20 strains), which were described later [4,5] or

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Abbreviations EIA, enzyme immunosorbent assay; GalA, galacturonic acid; LPS, lipopolysaccharide; PIH, passive immunohemolysis test; Qui3NAc, 3-acetamido-3,6-dideoxyglucose.

K. Zych et al. O-polysaccharides of Proteus genomospecies

recognized medically important

The 13C NMR spectrum of

species, two other P. penneri and P. hauseri, nor Proteus genomospecies 4, 5 and 6 [6–9]. Previously, as a result of the chemical and serological studies of LPS, a number of new Pro- teus serogroups were proposed for P. penneri strains [9,10]. Here we report on the structure of the O-specific polysaccharide from LPS of Proteus genomospecies 4 and propose to classify this strain into a new Proteus serogroup O56. We found also that Proteus genomo- species 5 and 6 should be classified into the existing Proteus serogroups O8 and O69, respectively.

Results and Discussion

Structural studies

the polysaccharide (Fig. 1) contained signals for five anomeric carbons at d 97.6, 98.1, 102.5, 104.0 and 104.3, three OCH2-C groups (C6 of Glc, GalN, and GlcN) at d 61.7, 62.3, and 69.9 [data of a distortionless enhancement of polari- zation transfer (DEPT) experiment], one CH3-C group (C6 of Qui3N) at d 19.8, one COOH group (C6 of GalA) at d 174.3, three nitrogen-bearing carbons (C2 of GlcN and GalN and C3 of Qui3N) at d 49.0–58.0, 17 oxygen-bearing ring carbons in the region d 68.8–81.8, and three N-acetyl groups at d 23.4–23.8 (CH3) and 175.1–176.0 (CO). Accordingly, the 1H NMR spectrum of the polysaccharide contained signals of equal inten- sity for five anomeric protons at d 4.52, 4.53, 4.74, 5.21 and 5.24 as well as signals for one CH3-C group (H6 of Qui3N) at d 1.36, three N-acetyl groups at d 1.98–2.05, and other signals at d 3.27–4.36. These data indicate that the polysaccharide has a regular structure with a pentasaccharide repeating unit.

The O-polysaccharide was obtained by mild acid degra- dation of the LPS, isolated from dried cells by the phenol ⁄ water procedure, and separated from lower molecular mass substances, including a core oligosac- charide, by Sephadex G-50 gel-permeation chromato- graphy. Sugar analyses by GLC of the alditol acetates derived after full acid hydrolysis of the polysaccharide revealed Glc, GlcN, GalN and 3-amino-3,6-dideoxyglu- cose (Qui3N) in the ratio 1 : 0.7 : 1 : 0.6. In addition, the acetylated methyl glycosides GLC analysis of showed the presence of GalA. GLC analyses of the acetylated (S)-2-(+)-butyl glycosides demonstrated the d configuration of Glc, GlcN, GalN, and GalA. The d configuration of Qui3N was established by the analysis of the 13C-NMR chemical shifts of the polysaccharide using the known regularities in glycosylation effects [11].

Methylation analysis of the polysaccharide, including GLC of partially methylated alditol acetates, resulted in the identification of the derivatives of terminal Glc, 3-substituted GalNAc, 6-substituted GlcNAc and 4-substituted Qui3NAc in the ratio 0.9 : 1 : 1 : 0.9. When the methylated polysaccharide was carboxyl- reduced prior to hydrolysis, 3-O-methylgalactose was identified in addition to the sugars mentioned above, which was evidently derived from 2,4-disubstituted GalA. Therefore, the O-polysaccharide has a branched pentasaccharide repeating unit containing terminal d-Glc, 3-substituted d-GalNAc, 6-substituted d-GlcNAc, 4-substituted d-Qui3NAc, and 2,4-disubstituted d-GalA.

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Fig. 1. 125-MHz 13C-NMR spectrum of the O-polysaccharide of Proteus genomospecies 4. Arabic numerals refer to carbon positions in sugar residues.

K. Zych et al. O-polysaccharides of Proteus genomospecies

Table 1. 1H- and 13C-NMR data of the polysaccharide of Proteus genomospecies 4 (d, p.p.m). The chemical shifts for the N-acetyl groups are dH 1.98, 2.04 and 2.05; dC 23.4, 23.6, 23.8 (all Me), 175.1, 175.3 and 176.0 (all CO).

Atom number

Sugar residue Nucleus 1 2 3 4 5 6 (6a,6b)

1H 13C 1H 13C 1H 13C 1H 13C 1H 13C

4.52 3.27 4.05 3.52 3.68 1.36 fi4)-b-D-Quip3NAc-(1fi 104.3 73.5 58.0 77.5 73.5 19.8 4.53 3.69 3.54 3.56 3.59 3.90, 4.17 fi6)-b-D-GlcpNAc-(1fi 102.5 56.6 74.9 70.7 75.7 69.9 4.74 3.54 3.71 3.80 3.85 fi2,4)-b-D-GalpA-(1fi 104.0 77.2 74.1 81.8 76.5 174.3 5.24 4.36 4.25 4.23 4.29 3.73 fi3)-a-D-GalpNAc-(1fi 97.6 49.0 77.5 68.8 72.4 62.3 5.21 3.53 3.74 3.45 4.20 3.66, 3.87 a-D-Glcp-(1fi 98.1 72.9 74.1 70.6 73.0 61.7

(d

The 1H- and 13C-NMR spectra of the O-polysaccha- ride were assigned using 2D COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments (Table 1). The spin systems for GalA and Qui3NAc were identified by correlations of H1 with H2-H5 and H2-H6, respectively, in the TOCSY spectrum and with H3,H5 in the ROESY spectrum. Correlations of H1 with H2-H6 in the TOCSY spectrum demonstrated Glc and GlcNAc. GalNAc showed correlations for H1 with H2-H4 in the TOCSY spectrum and H4 with H5,H6 in the ROESY spectrum. The carbonyl signal of GalA was distinguished by an H5 ⁄ C6 correlation in the HMBC spectrum.

J1,2 coupling constant values of (cid:1) 3 Hz indicated that GalNAc and Glc are a-linked, whereas J1,2-values of 7–8 Hz showed that Qui3NAc, GlcNAc, and GalA are b-linked. The pyranose form of all monosaccharide from the residues originated from the

absence

Qui3NAc H1 ⁄ GlcNAc H6a and H6b (d 4.52 ⁄ 3.90 and 4.17); GlcNAc H1 ⁄ GalA H4 (d 4.53 ⁄ 3.80); GalA H1 ⁄ GalNAc H3 4.74 ⁄ 4.25); GalNAc H1 ⁄ Qui3NAc H4 (d 5.24 ⁄ 3.52); and Glc H1 ⁄ GalA H2 (d 5.21 ⁄ 3.54) correlations. In accordance with the the HMBC experiment showed the ROESY data, following inter-residue cross-peaks between the atoms separated by three bonds: Qui3NAc H1 ⁄ GlcNAc C6 (d 4.52 ⁄ 69.9); GlcNAc H1 ⁄ GalA C4 (d 4.53 ⁄ 81.8); GalA H1 ⁄ GalNAc C3 (d 4.74 ⁄ 77.5); GalNAc C1 ⁄ Qui3- NAc H4 (d 97.6 ⁄ 3.52) and Glc C1 ⁄ GalA H2 (d 98.1 ⁄ 3.54). These data were in agreement also with the glycosylation pattern determined from methylation and 13C-NMR chemical shift data (see above) and defined the monosaccharide sequence in the repeating unit. Therefore, it was concluded that the O-polysaccharide of Proteus genomospecies 4 has the structure:

13C-NMR spectrum of any signals for nonanomeric ring carbons at a field lower than d 82 [12].

The O-polysaccharide has a unique structure among Proteus polysaccharides and is distinguished by the presence of 3-acetamido-3,6-dideoxy-d-glucose. While this is a rarely occurring component, it was found in several other bacterial polysaccharides [15], including O-polysaccharides of Proteus [16].

A relatively low-field position of the signals for C6 of GlcNAc (d 69.9), C3 of GalNAc (d 77.5), C4 of Qui3NAc (d 77.5) as well as C2 and C4 of GalA (d 77.2 and 81.8, respectively), as compared with their position in the corresponding nonsubstituted monosac- charides [13,14], demonstrated the modes of glycosyla- tion of the constituent sugar residues.

Studies of the O-polysaccharides of Proteus genomo- species 5 and 6 and the comparison of the correspond- ing 13C NMR spectra indicated that they are identical to those of P. vulgaris O8 [17] and P. penneri 25 (O69) [18], respectively.

A ROESY experiment (Fig. 2) revealed interresidue cross-peaks between the anomeric protons and glyco- sidically linked protons, which were assigned to

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K. Zych et al. O-polysaccharides of Proteus genomospecies

Fig. 2. Part of a 2D ROESY spectrum of the polysaccharide of Proteus genomospecies 4. The corresponding parts of the 1H-NMR spectrum are displayed along the axes. Ara- bic numerals refer to proton positions in sugar residues.

in the

tyl group at d 23.6 (CH3; 23.7 in P. penneri 25) and 175.7 (CO; 175.7 in P. penneri 25), 14 other oxygen- bearing ring carbons region d 69.6–84.0 (d 69.7–84.2 in P. penneri 25) as well as signals for alanine at d 172.5, 50.8 and 17.7 (d 172.5, 50.8 and 17.6 in P. penneri 25).

Serological studies

In particular, the 13C NMR spectrum of Proteus genomospecies 5 contained signals for four anomeric carbons at d 99.3, 99.8, 102.4 and 105.1 (d 99.6, 100.0, 102.5 and 105.3 in P. vulgaris O8), two OCH2-C groups at d 62.2 and 63.3 (d 62.4 and 63.4 in P. vul- garis O8), two nitrogen-bearing carbons at d 50.4 and 55.4 (d 50.6 and 55.5 in P. vulgaris O8), one CH3-C group at d 16.6 (d 16.7 in P. vulgaris O8), one COOH group at d (d 173.5 in P. vulgaris O8), two N-acetyl groups at d 23.5 and 23.8 (CH3; 23.7 and 23.9 in P. vulgaris O8) and 176.1 and 176.2 (CO; 176.2 and 176.3 in P. vulgaris O8) as well as 12 other oxygen- bearing ring carbons in the region d 69.6–84.0 (d 69.7– 84.2 in P. vulgaris O8).

Serological investigations were performed using LPS- specific rabbit polyclonal antisera against strains of Proteus genomospecies 4, 5 and 6. They were tested with LPS of a number of Proteus strains with known O-polysaccharide structure in passive immunohemoly- sis (PIH). In addition, LPS of Providencia stuartii O18 was investigated in order to confirm the serological relatedness between this bacterium and Proteus genomospecies 4 reported earlier [19].

From 92 tested Proteus lipopolysaccharides,

Similarly, the 13C NMR spectrum of the O-deacetyl- ated polysaccharide of Proteus genomospecies 6 con- tained signals for four anomeric carbons at d 102.2, 102.3, 102.9 and 104.1 (d 101.9, 102.0, 102.7 and 104.0 in P. penneri 25), two OCH2-C groups at d 61.8 and 68.7 (d 61.9 and 68.8 in P. penneri 25), two nitrogen- bearing carbons at d 55.4 and 56.1 (d 55.3 and 56.0 in P. penneri 25), two COOH groups at d 174.6 and 175.7 (d 174.9 and 176.0 in P. penneri 25), one N-ace-

inclu- ding those of 27 strains of P. vulgaris, 39 strains of P. mirabilis, 24 strains of P. penneri, one strain of P. myxofaciens, one strain of P. hauseri and Providen- cia stuartii O18, only LPS of P. vulgaris OX2 and

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K. Zych et al. O-polysaccharides of Proteus genomospecies

OX2 removed antibodies to the corresponding cross- reactive LPS but influenced the reactivity with the homologous LPS insignificantly. Hence, cross-reactive is only a minor population of antibodies.

In western blot (SDS ⁄ PAGE separation of the investi- gated LPSs also presented on Fig. 4A), Proteus genomospecies 4 antiserum (Fig. 4. B) strongly reacted with slow migrating bands of the homologous LPS and that of Providencia stuartii O18. These and absorption data suggest that the antiserum contains both major O-polysaccharide-specific antibodies, which recognize an epitope on the homologous O-antigen, and a minor population of antibodies, which react with a common epitope on the O-antigen of Proteus genomospecies 4 and Providencia stuartii O18. In addition, Proteus ge- nomospecies 4 antiserum recognized fast migrating LPS bands of the two bacteria as well as those of P. vulgaris OX2; these correspond to the LPS core with no O-poly- saccharide attached. Therefore, there are also LPS core-specific cross-reactive antibodies, which recognize different epitopes on the Providencia stuartii O18 and P. vulgaris OX2 LPS, as followed from combined western blot and absorption data.

fragment

Fig. 3. EIA binding curves of Proteus genomospecies 4 (A), 5 (B) and 6 (C) antisera to the investigated LPS.

chain (Fig. 5). Moreover,

(Fig. 3A)

Serological relatedness of Proteus genomospecies 4 and Providencia stuartii O18 demonstrated in this work is in agreement with the cross-reactivity of LPS the Proteus strain with anti-Providencia stuartii of O18 serum [19]. Comparison of the O-polysaccharide structures of Proteus genomospecies 4 and Providencia stuartii O18 showed that they share a b-d-Quip3NAc- (1 fi 6)-d-GlcNAc (a), disaccharide which, most likely, occupies the nonreducing end of the polysaccharide the O-polysaccharides possess also a common interior a-d-GalpNAc-(1 fi 4)-b-d-Quip3NAc-(1 fi 6)-d-GlcNAc trisaccharides fragment (b), and both common features seem to be responsible for the cross-reactivity of these strains. Proteus vulgaris OX2 has no similarities with Proteus genomospecies 4 in O-antigen structure [20], which is consistent with the western blot data showing the absence of any common epitope on the O-polysac- charides (Fig. 4B).

Providencia stuartii O18 cross-reacted with Proteus genomospecies 4 antiserum. As opposed to the reac- tion of Proteus genomospecies 4 antiserum with the homologous LPS (1 : 51 200), the cross-reactions were rather weak (1 : 1600 and 1 : 6400, respectively). Their specificity was confirmed using enzyme immunosorbent assay (EIA) (1 : 256 000, 1 : 8000 and 1 : 32 000, respectively) and inhibition of the reaction in both PIH and EIA. Again, the homologous LPS was a strong inhibitor (1–4 ng) whereas the inhibiting activity of the cross-reactive antigens was significantly weaker (1250–5000 ng).

The antigens

Based on the results of serological studies and the uniqueness of the O-polysaccharide structure we pro- pose classifying Proteus genomospecies 4 into a new Proteus serogroup, O56, in which at present this strain is the single representative.

genomospecies

studied were tested in PIH with Proteus antiserum, which was 4 absorbed with the respective LPS. The reactivity of antiserum against Proteus genomospecies 4 with all tested antigens was completely abolished when it was absorbed with the homologous LPS. Absorption with LPS of either Providencia stuartii O18 or P. vulgaris

In PIH and EIA LPS from P. vulgaris O8 cross- reacted with Proteus genomospecies 5 antiserum (Fig. 3B) and that from P. penneri O69 with Proteus genomospecies 6 antiserum (Fig. 3C), the cross-reac- tions being almost identical to the reactions of the homologous LPS (1 : 51 200). Inhibition of the reac-

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K. Zych et al. O-polysaccharides of Proteus genomospecies

A

B

C

D

Fig. 4. SDS ⁄ PAGE (A) and western blot of Proteus genomospecies 4 (B), 5 (C) and 6 (D) antisera with LPS from investigated strains. Abbre- viations: P.s., Providencia stuartii; P.g., Proteus genomospecies; P.p., P. penneri; P.v., P. vulgaris. The antisera were diluted in blotting buffer (1 : 300); 3–5 ng LPS were applied per lane. Concentration of acrylamide in the gel was 12%.

tions in both PIH and EIA was also very strong and similar (1–2 ng), indicating a high level of serological relatedness between the two pairs of bacteria. The reactivity of both Proteus genomospecies 5 and 6 anti- sera was completely abolished by absorption with the respective homologous and cross-reactive antigens. In western blot, Proteus genomospecies 5 and 6 antisera (Fig. 4C,D) reacted similarly with both homologous and heterologous P. vulgaris O8 and P. penneri O69 LPS, respectively. The reactions were strong with slow migrating LPS bands and weaker with fast migrating bands, which correspond to the LPS core with and without the O-polysaccharide, respectively.

structures

Fig. 5. Structures of the O-polysaccharides of cross-reactive bac- teria Proteus genomospecies 4 (this work), Providencia stuartii O18 [19] and P. vulgaris OX2 [20].

The serological data obtained and the identity of the showed that Proteus O-polysaccharide genomospecies 5 and 6 strains should be classified into the existing Proteus serogroups O8 and O69 [17,18,21], respectively.

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K. Zych et al. O-polysaccharides of Proteus genomospecies

Experimental procedures

Bacterial strains and growth

4

5

genomospecies

(ATCC 51469),

NaBH4 in 1 m ammonia (20 (cid:1)C, 1 h), acetylated with a 1 : 1 (v ⁄ v) mixture of pyridine and acetic anhydride (120 (cid:1)C, 30 min) and analysed by GLC, using a Hewlett- Packard 5890 Series II instrument, equipped with a DB- 225 capillary column (30 m · 0.25 mm) and a temperature gradient 170–180 (cid:1)C at 1 (cid:1)CÆmin)1 and then 180–230 (cid:1)C at 7 (cid:1)CÆmin)1. Methanolysis of the polysaccharide (1 mg) was performed with 1 m HCl in MeOH (85 (cid:1)C, 16 h) and was followed by acetylation and GLC analysis as des- cribed above. The absolute configuration of the mono- saccharides was determined by GLC of the acetylated (S)-(+)-2-butyl glycosides according to the published method [25].

Methylation analysis

Proteus (ATCC 51470), 6 (ATCC 51471), P. hauseri (ATCC 700826) and 24 P. penneri strains were kindly provided by C. M. O’Hara and D. J. Brenner (Centres for Disease Control and Preven- tion, Atlanta, GA, USA). Thirty-nine strains of P. mirabilis and 27 P. vulgaris strains were from the Czech National Collection of Type Cultures (CNCTC, National Institute of Public Health, Prague, Czech Republic). Proteus myxofac- iens strain (CCUG 18769) was kindly provided by E. Fal- sen [Cultures Collection, University of Goeteborg (CCUG), Goeteborg, Sweden] and Providencia stuartii O18 was from the Hungarian National Collection of Medical Bacteria (National Institute of Hygiene, Budapest, Hungary). Dry bacterial mass was obtained from aerated culture as des- cribed previously [22].

Isolation and degradation of lipopolysaccharide

Methylation of the polysaccharide was performed with CH3I in dimethylsulfoxide in the presence of sodium meth- ylsulfinylmethanide [26]. A portion of the methylated poly- saccharide was reduced with LiBH4 in 70% (v ⁄ v) aqueous 2-propanol (20 (cid:1)C, 16 h). Partially methylated monosaccha- rides were derived by hydrolysis under the same conditions as in sugar analysis, reduced with NaBH4 or NaBD4, acet- ylated and analysed by GLC-MS using a Trace GC 2000 instrument and a temperature gradient 150–250 (cid:1)C at 10 (cid:1)CÆmin)1.

Lipopolysaccharides were obtained by extraction of bacter- ial mass with a hot phenol ⁄ water mixture [23] and purified by treatment with aqueous 50% (v ⁄ v) CCl3CO2H at 4 (cid:1)C followed by dialysis of the supernatant. Alkali-treated LPS were prepared by saponification of LPS with 0.25 m NaOH (56 (cid:1)C, 2 h) followed by precipitation with ethanol.

NMR spectroscopy

internal

(dH 2.225 p.p.m.,

Acid degradation of Proteus genomospecies 4 LPS (226 mg) was performed with aqueous 2% HOAc at 100 (cid:1)C until lipid A precipitation. The precipitate was removed by centrifugation (13 000 g, 20 min), and the supernatant was fractionated on a column (56 · 2.6 cm) of Sephadex G-50 (Pharmacia, Fairfield, CT, USA) in 0.05 m pyridinium acet- ate buffer, pH 4.5, monitored using a Knauer differential refractometer (Berlin, Germany). A high-molecular mass polysaccharide was obtained in a yield of (cid:1) 10% of the LPS weight. All experiments followed 36 ⁄ 609 EEC guidelines.

NMR spectra were recorded on a Bruker DRX-500 spec- trometer (Karlsruhe, Germany) for a solution in D2O at dC 40 (cid:1)C using acetone 31.45 p.p.m.) as reference. Prior to the measurements, sam- ples were deuterium-exchanged by freeze-drying twice from D2O. Bruker software xwinnmr 2.1 was used to acquire and process the NMR data. A mixing time of 200 and 300 ms was used in two-dimensional TOCSY and ROESY experiments, respectively. The other parameters used for 2D experiments were essentially the same as described previously [27].

Rabbit antiserum and serological assays

Acknowledgements

The work was supported by the following grants: Uni- versity of Lodz (grant 801), 02-04-48767 of the Russian Foundation for Basic Research, RFMK-226.2003.03 and RF State Contract RI–112 ⁄ 001 ⁄ 272.

Polyclonal LPS-specific antisera were obtained in a stand- ard procedures by immunization of New Zealand white rabbits (two for one bacterial strain) with heat-inactivated (100 (cid:1)C, 2,5 h) bacteria of Proteus genomospecies 4, 5 and 6, as described previously [24]. Passive immunohemolysis, absorption, EIA and inhibition experiments as well as SDS ⁄ PAGE and western blot were carried out as described [24]. Lipopolysaccharide (EIA) or alkali-treated LPS (PIH) were used as antigens.

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