Structure of the O-polysaccharide leads to classification of Proteus penneri 31 in Proteus serogroup O19

FEMS Immunology and Medical Microbiology 39 (2003) 73^79

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Structure of the O-polysaccharide leads to classi¢cation of Proteus penneri 31 in Proteus serogroup O19 Anna N. Kondakova a , Krystyna Zych b , Sof’ya N. Senchenkova a , Agnieszka ZabIotni b , Alexander S. Shashkov a , Yuriy A. Knirel a , Zygmunt Sidorczyk b; b

a N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia Department of General Microbiology, Institute of Microbiology and Immunology, University of LIo¤dz¤, Banacha 12/16, 90-237 LIo¤dz¤, Poland

Received 26 April 2003; received in revised form 18 June 2003; accepted 23 June 2003 First published online 15 July 2003

Abstract O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide (LPS) of Proteus penneri strain 31. Sugar and methylation analyses along with NMR spectroscopic studies, including 2D 1 H,1 H COSY, TOCSY, ROESY, 1 H,13 C and 1 H,31 P HMQC experiments, demonstrated the following structure of the polysaccharide:

where FucNAc is 2-acetamido-2,6-dideoxygalactose and EtnP is 2-aminoethyl phosphate. The polysaccharide studied has the same carbohydrate backbone as the O-polysaccharide of Proteus vulgaris O19. Based on this finding and close serological relatedness of the LPS of the two strains, it is proposed to classify P. penneri 31 in Proteus serogroup O19 as an additional subgroup. In contrast, D-GlcNAc6PEtn and K-L-FucNAc-(1C3)-D-GlcNAc shared with a number of other Proteus O-polysaccharides could not provide any significant cross-reactivity of the corresponding LPS with rabbit polyclonal O-antiserum against P. penneri 31. @ 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Proteus penneri ; O-antigen; O-serogroup; Lipopolysaccharide ; Serological classi¢cation

1. Introduction Being common in human and animal intestines, under favourable conditions Gram-negative bacteria of the genus Proteus from the family Enterobacteriaceae cause various infections. From them, most important are urinary tract infections, which can lead to severe complications, such as acute or chronic pyelonephritis and formation of bladder

* Corresponding author. Tel. : +48 (42) 635 44 67; Fax : +48 (42) 678 49 32. E-mail address : [email protected] (Z. Sidorczyk). Abbreviations : EIA, enzyme immunosorbent assay; EtnP, 2-aminoethyl phosphate ; LPS, lipopolysaccharide; FucNAc, 2-acetamido-2,6dideoxygalactose (N-acetylfucosamine); PI, passive immunohemolysis

and kidney stones. After Proteus mirabilis and Proteus vulgaris, Proteus penneri is the third distinguished species of these medically important bacteria. As opposite to the ¢rst two species, serological classi¢cation of P. penneri is far from being complete. Recently, on the basis of immunochemical studies of the lipopolysaccharide (LPS), a number of new O-serogroups have been proposed for P. penneri strains [1]. O-polysaccharides of most P. penneri strains studied are acidic due to the presence of uronic acids and various acidic non-sugar components, such as amino acids, lactic and pyruvic acids and phosphate groups [2^5]. Now we report on the structure of another acidic O-polysaccharide isolated from P. penneri strain 31, which shares some structural features with the O-polysaccharides of several other Proteus strains studied by us earlier. Serological relatedness of the LPS of some of these strains was revealed

0928-8244 / 03 / $22.00 @ 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0928-8244(03)00205-0

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and served as the basis for classi¢cation of P. penneri 31 in Proteus serogroup O19.

2. Materials and methods 2.1. Bacterial strains, growth, isolation and degradation of the LPS P. penneri strains 16 (CDC 465-75), 20 (CDC 0766-80), 28 (ITJ 339) and 31 (TGH 629, isolated from urine of a patient with bacteriuria in Toronto, Canada) were kindly provided by Prof. D.J. Brenner (Center for Diseases Control, Atlanta, GA, USA). Strains of P. vulgaris and P. mirabilis were from the Czech National Collection of Type Cultures (CNCTC, Institute of Microbiology and Genetics, Prague, Czech Republic). Dry bacterial cells of P. penneri 31 were obtained from aerated liquid culture as described [6]. LPS was obtained by extraction of dried bacterial cells with a hot phenol^water mixture [7] and puri¢ed by treatment with cold aqueous 50% CCl3 CO2 H followed by dialysis of the supernatant [8]. Degradation of LPS was performed with aqueous 1% HOAc at 100‡C for 2 h, and products were fractionated by gel-permeation chromatography on a column (3U65 cm) of Sephadex G-50 in 0.05 M pyridinium acetate bu¡er (pH 4.5) monitored using a Knauer di¡erential refractometer (Germany) to give a high-molecular-mass polysaccharide.

(+)-2-octyl glycosides according to the published method [14] modi¢ed as described [15]. Methylation was performed according to the Hakomori procedure [16]. Prior to methylation, the polysaccharide was dephosphorylated with aqueous 48% HF (4‡C, 16 h). The 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 Hewlett-Packard 5890 chromatograph equipped with a NERMAG R10-10L mass spectrometer (France) under the same chromatographic conditions as in sugar analysis. 2.4. Nuclear magnetic resonance (NMR) spectroscopy 1

H, 13 C, and 31 P NMR spectra were recorded with a Bruker DRX-500 spectrometer in D2 O at 60‡C using internal acetone (NH 2.225, Nc 31.45) or external aqueous 85% H3 PO4 (NP 0) as reference. 2D NMR spectra were obtained using standard Bruker software, and the XWINNMR 2.1 program (Bruker) was used to acquire and process the data. A mixing time of 150 and 300 ms was used in 2D TOCSY and ROESY experiments, respectively. Other parameters used for 2D experiments were essentially the same as described previously [17].

3. Results and discussion 3.1. Structural studies of the O-polysaccharide

2.2. Rabbit antiserum and serological assays Polyclonal O-antiserum was obtained by immunisation of rabbits with heat-inactivated bacteria of P. penneri 31 according to the published procedure [9]. SDS^PAGE, electrotransfer of LPS from gels to nitrocellulose sheets, immunostaining and absorption experiments were carried out as described [10]. LPS was used as antigen in enzyme immunosorbent assay (EIA) [11]. Passive immunohemolysis (PI) was performed with increasing amounts (2^200 Wg) of alkali-treated LPS [11]. 2.3. Sugar and methylation analyses The polysaccharide was hydrolysed with 2 M CF3 CO2 H (120‡C, 3 h). Amino components were identi¢ed using a Biotronik LC-2000 amino acid analyser equipped with a column (22U0.4 cm) of Ostion LG AN B cation-exchange resin in 0.2 M sodium citrate bu¡er (pH 3.25) at 65‡C. Neutral sugars were analysed using a Biotronik LC-2000 sugar analyser as described [12]. The alditol acetates were derived as described [13] and analysed by gas-liquid chromatography (GLC) using a Hewlett-Packard 5890 chromatograph (USA) equipped with a DB-5 fused-silica capillary column. The absolute con¢gurations of the monosaccharides were determined by GLC of acetylated

The O-polysaccharide was obtained by mild acid degradation of the LPS of P. penneri 31 followed by gel chromatography on Sephadex G-50. Chemical analyses using amino acid and sugar analysers after hydrolysis of the polysaccharide revealed galactose and four amino components: 2-amino-2-deoxyglucose, 2-amino-2-deoxygalactose, 2-amino-2,6-dideoxygalactose (FucN) and 2-aminoethanol in almost equal amounts. The identi¢cation of the monosaccharides was additionally con¢rmed by GLC of the alditol acetates and acetylated (+)-2-octyl glycosides, which showed the L con¢guration of FucN and the D con¢guration of the other monosaccharides. The 13 C-NMR spectrum of the polysaccharide demonstrated a tetrasaccharide repeating unit. The spectrum showed signals for four anomeric carbons at N 98.7^ 103.9, two unsubstituted HOCH2 -C groups (C6) at N 61.6 and 61.9, one O-substituted HOCH2 -C group at N 65.5 (data of attached-proton test [18]), one CH3 -C group at N 16.2 (C6 of FucN), three nitrogen-bearing carbons at N 49.6, 51.2 and 56.8 (C2 of amino sugars), 13 sugar ring oxygen-bearing carbons in the region N 67.8^80.7, three N-acetyl group (CH3 at N 23.4^23.5, CO at N 174.8^ 175.3) and one residue of 2-aminoethanol (CH2 N at N 41.5 and CH2 O at N 62.1). Accordingly, the 1 H-NMR spectrum of the polysaccha-

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Table 1 1 H NMR data of the O-polysaccharide of P. penneri 31 (N, ppm) Residue

H1

H2

H3

H4

H5

H6a

H6b

C3)-K-D-Galp-(1C C4)-K-D-GalpNAc-(1C C3)-K-L-FucpNAc-(1C C3)-L-D-GlcpNAc-(1C Etn

4.93 5.12 5.01 4.67 4.12

3.90 4.21 4.32 3.92 3.28

3.95 4.01 4.02 3.73

4.23 4.09 3.91 3.64

4.03 4.29 4.42 3.58

3.82 3.64 1.19 4.17

3.89 3.71 4.17

Chemical shifts for NAc are N 1.96, 1.98 and 2.03.

ride contained signals for four anomeric protons at N 4.67^ 5.12, one CH3 -C group at N 1.19 (doublet, J5;6 6.26 Hz, H6 of FucN), three N-acetyl groups at N 1.96^2.03 (all singlets) and one residue of 2-aminoethanol (CH2 N at N 3.28, triplet, J 5 Hz). The 31 P-NMR spectrum of the polysaccharide contained one signal for a monophosphate group at N 3.2. Therefore, the polysaccharide has a tetrasaccharide repeating unit that contains one residue each of D-Gal, D-GlcNAc, D-GalNAc, L-FucNAc and 2-aminoethyl phosphate (EtnP). Methylation analysis using GLC-MS of the partially methylated alditol acetates derived from the dephosphorylated polysaccharide demonstrated 3-substituted Gal, GlcNAc and FucNAc, and 4-substituted GalNAc. Therefore, the polysaccharide is linear. The 1 H- and 13 C-NMR spectra were assigned using 2D shift-correlated experiments, including 1 H,1 H COSY, TOCSY, ROESY and H-detected 1 H,13 C HMQC, and the chemical shifts are tabulated in Tables 1 and 2. The sugar residues were identi¢ed as GlcpNAc (J3;4 and J4;5 V10 Hz), GalpNAc, FucpNAc and Galp (J3;4 and J4;5 9 3 Hz). FucpNAc was recognised by a H5/H6 correlation at N 4.42/1.19 in the COSY spectrum and a H5/H4 correlation at N 4.42/3.91 in the ROESY spectrum, as well as by a low-¢eld position of the signal for H2 at N 4.32 and its correlation to the signal for C2 at N 49.6 in the 1 H,13 C HMQC spectrum. Similarly, the spin systems of GlcpNAc and GalpNAc were distinguished by correlation of the signals for the protons at nitrogen-bearing carbons (H2) to the corresponding carbons (C2) at N 56.8 and 51.2. As judged by a relatively large J1;2 coupling constant value of 7^8 Hz for the H1 signal at N 4.67, GlcNAc is L-linked. The signals for the three remaining anomeric protons were not clearly resolved; their relatively low-¢eld position at N 4.93^5.12 enabled suggestion that they are K-linked. The con¢gurations of the glycosidic linkages

were con¢rmed by the ROESY spectrum, which showed correlations between H1 and H3,5 for the L-linked GlcNAc and between H1 and H2 for the remaining, K-linked sugar residues. The 1 H,31 P HMQC experiment showed correlations of the phosphorus signal at N 3.2 with the proton signals of the 2-aminoethanol group at N 3.28 (CH2 N) and 4.12 (CH2 O), as well as with the H6 signal of GlcNAc at N 4.17. These data indicated that EtnP is attached at position 6 of GlcNAc, which was con¢rmed by a low-¢eld position of the signal for C6 of GlcNAc at N 65.5 (K-e¡ect of phosphorylation; compare the chemical shift N 61.9 for C6 in the non-substituted monosaccharide [21]). Low-¢eld displacements in the 13 C-NMR spectrum of the signals for C3 of Gal, GlcNAc and FucNAc and C4 of GalNAc by 4^11 ppm (K-e¡ect of glycosylation), as compared with their positions in the spectra of the corresponding unsubstituted monosaccharides [19], revealed the glycosylation pattern of the monosaccharide residues in the repeating unit, which was in agreement with the methylation analysis data (see above). In addition to intraresidue NOE correlations, the ROESY spectrum of the polysaccharide showed the following interresidue correlations between the anomeric protons and protons at the linkage carbons : Gal H1/GalNAc H4 at N 4.93/4.09; GalNAc H1/FucNAc H3 at N 5.12/4.02; FucNAc H1/GlcNAc H3 at N 5.01/3.73; and GlcNAc H1/Gal H3 at N 4.67/3.95. These data are in agreement with the glycosylation pattern and de¢ned the full sequence of the monosaccharide residues in the repeating unit. Therefore, the O-polysaccharide of P. penneri 31 has the structure 1 shown in Fig. 1. This polysaccharide has some structural features in common with several other Proteus O-polysaccharides. For instance, L-D-GlcNAc6PEtn has been found in the O-polysaccharides of P. mirabilis O27 [20], P. penneri 8

Table 2 13 C NMR data of the O-polysaccharide of P. penneri 31 (N, ppm) Residue

C1

C2

C3

C4

C5

C6

C3)-K-D-Galp-(1C C4)-K-D-GalpNAc-(1C C3)-K-L-FucpNAc-(1C C3)-L-D-GlcpNAc-(1C Etn

101.8 99.6 98.7 103.9 62.1

68.6 51.2 49.6 56.8 41.5

80.7 68.3 74.4 79.0

69.7 79.4 72.1 69.0

73.0 71.9 67.8 75.4

61.9 61.6 16.2 65.5

Chemical shifts for NAc are N 23.4, 23.4 and 23.5 (all Me), 174.8, 175.0 and 175.3 (all CO).

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Fig. 1. Structures of the O-polysaccharides of P. penneri 31 (1) (this work), P. vulgaris O19 (2) [23], P. penneri 16 and 20 (3) [24] and P. penneri 28 (4) [25]. D-Fuc3N(R-3HOBu) stands for 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-D-galactose and D-GlcNAc3(S-Lac) for 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (N-acetylisomuramic acid).

(O67) [21] and P. penneri 63 (O68) [22]. Furthermore, an K-L-FucNAc-(1C3)-D-GlcNAc fragment is present in all Proteus polysaccharides that contain FucNAc [2,21^23]. Most remarkably, the O-polysaccharide of P. penneri 31 has the same carbohydrate backbone as the O-polysaccharide of P. vulgaris O19 [23] (structure 2 in Fig. 1), from which it di¡ers in the presence of EtnP only.

inhibition experiments in the system P. penneri 31 O-antiserum/P. penneri 31 LPS using PI and EIA (Table 3). The LPS of P. penneri 31 and P. vulgaris O19 strongly inhibited the reaction, whereas the other heterologous LPS were signi¢cantly weaker inhibitors. In Western blot (Fig. 2A), P. penneri 31 O-antiserum reacted with slow migrating bands of the homologous LPS and the LPS of P. vulgaris O19, P. penneri 16 and 20 as well as with fast migrating bands of all tested antigens. The slow and fast migrating bands correspond to the LPS species consisting of a core-lipid A moiety with or without a O-polysaccharide chain attached to the core, respectively. The slow migrating band pattern of the O-polysaccharide-containing LPS species of P. penneri 31 LPS was similar to that of P. vulgaris O19 and di¡erent from those of P. penneri 16 and 20. The fast migrating band patterns were similar for P. penneri 16, 28 and 31 and di¡erent for P. penneri 20 and P. vulgaris O19. Absorption of P. penneri 31 O-antiserum with the LPS of P. penneri 28 removed antibodies that recognise both O-polysaccharide and core epitopes in the LPS of P. penneri 16, 20 and 28 but did not a¡ect binding pattern of the LPS of P. penneri 31 and P. vulgaris O19 (data not shown). The reactivity of P. penneri 31 O-antiserum with all

3.2. Serological studies of the LPS From the LPS of 130 Proteus strains tested, only ¢ve reacted with rabbit polyclonal P. penneri 31 O-antiserum in PI. These were the homologous LPS and those of P. penneri 16, 20, 28 and P. vulgaris O19 (Table 3; for the O-antigen structures of these strains [23^25] see Fig. 1). The last strain reacted as strong as the homologous LPS, whereas the reactivity of P. penneri 28 was slightly weaker and that P. penneri 16 and 20 much weaker. Similar results were obtained in EIA (Table 3). Almost no cross-reaction was observed with the LPS of P. mirabilis O27, P. penneri 8 (O67) and P. penneri 63 (O68), which share with P. penneri 31 a L-D-GlcNAc6PEtn residue (all three strains) and an K-L-FucNAc-(1C3)-D-GlcNAc disaccharide (P. penneri 8 and P. penneri 63) [20^22]. The speci¢city of the cross-reactions was con¢rmed by

Table 3 Reactivity of O-antisera against P. penneri 31 and P. vulgaris O19 with the Proteus LPS LPS from Proteus strain

P. P. P. P. P. P. P. P. P.

penneri 31 O-antiserum penneri 31 vulgaris O19 penneri 16 penneri 20 penneri 28 vulgaris O19 O-antiserum penneri 31 vulgaris O19

Reciprocal titre for the LPS in

Minimal inhibiting dose (ng) of the LPS in

EIA

EIA

PI

512 000 128 000 32 000 32 000 64 000

25 600 25 600 6 400 3 200 12 800

128 000 128 000

12 800 51 200

PI

P. penneri 31 O-antiserum/P. penneri 31 LPS 15.6 2 31.2 2 5000 625 5000 625 5000 625 P. vulgaris O19 O-antiserum/P. vulgaris O19 LPS 2 2 1 1

LPS and alkali-treated LPS were used as antigen in EIA and PI, respectively. The data of the homologous LPS are shown in bold type.

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tested antigens in PI was completely abolished when it was absorbed with the homologous LPS (Table 4). Absorption with the LPS of P. vulgaris O19 removed antibodies to this LPS, decreased the reactivity with the P. penneri 31 LPS and did not in£uence the reaction with P. penneri 16, 20 and 28. Absorption with each of the last three LPS had no in£uence on the reaction with the P. vulgaris O19 LPS, slightly decreased the reactivity with the P. penneri 31 LPS and completely abolished that with the three other antigens. Double absorption ¢rst with the P. penneri 28 LPS to remove anti-core antibodies and then with the P. vulgaris O19 LPS to remove antibodies against a common Opolysaccharide epitope(s) decreased the reactivity with the homologous LPS and completely abolished the reactivity with all heterologous antigens. The remaining antibodies are thus directed against an epitope(s) on the O-polysaccharide of P. penneri 31 that is absent from P. vulgaris O19. Studies with P. vulgaris O19 O-antiserum using PI and EIA as well as inhibition experiments (Table 3) con¢rmed close serological relatedness between P. vulgaris O19 and P. penneri 31. Western blot (Fig. 2B) showed a similar binding pattern of P. vulgaris O19 O-antiserum to slow migrating LPS bands of both strains and a di¡erent binding pattern to fast migrating bands. These data suggest similarity in O-polysaccharide epitopes and both similarity and di¡erences in core epitopes of P. vulgaris O19 and P. penneri 31. Absorption of P. vulgaris O19 O-antiserum with the homologous LPS completely abolished the reactivity with both antigens, whereas absorption with the P. penneri 31 LPS kept minor antibodies reactive with the homologous LPS (Table 4), which, most likely, are core-speci¢c. P. vulgaris O19 O-antiserum did not crossreact with the LPS of P. penneri 16, 20 and 28 in any of the assays. As mentioned above, the O-polysaccharide of P. penneri 31 has the same carbohydrate backbone as that of

77

Fig. 2. Western blot of Proteus LPS with O-antisera against P. penneri 31 (A) and P. vulgaris O19 (B). P.p. and P.v. stand for P. penneri and P. vulgaris, respectively.

Table 4 PI of the alkali-treated Proteus LPS with absorbed O-antisera against P. penneri 31 and P. vulgaris O19 O-antisera absorbed with the alkali-treated LPS from Proteus strain

P. penneri 31 O-antiserum Control P. penneri 31 P. vulgaris O19 P. penneri 16 P. penneri 20 P. penneri 28 P. penneri 28 and P. vulgaris O19 P. vulgaris O19 O-antiserum Control P. vulgaris O19 P. penneri 31

Reciprocal titre of absorbed O-antisera for the alkali-treated LPS from Proteus strain P. penneri 31

P. vulgaris O19

25 600 6 100 6 400 12 800 12 800 12 800 1 600

25 600 6 100 6 100 25 600 25 600 25 600 6 100

12 800 6 100 6 100

51 200 6 100 3 200

Sheep red blood cells were used as control.

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P. penneri 16 6400 6 100 6400 6 100 6 100 6 100 6 100

P. penneri 20

P. penneri 28

3200 6 100 3200 6 100 6 100 6 100 6 100

6400 6 100 6400 6 100 6 100 6 100 6 100

78

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P. vulgaris O19 [23], the two antigens di¡ering only in the absence of EtnP from the latter (Fig. 1). The structural and serological data of the LPS suggest that it is reasonable to classify P. penneri 31 to Proteus O19 serogroup and divide this serogroup to two subgroups : O19a for P. vulgaris O19 and O19a,19b for P. penneri 31. Factor O19a is linked to a common epitope on the O-polysaccharides and factor O19b to a particular epitope that is present on the O-polysaccharide of P. penneri 31 only and is evidently associated with EtnP. O19b seems to be a minor epitope since sharing by the O-polysaccharides of not only EtnP but also GlcNAc6PEtn could not provide any signi¢cant cross-reactivity of the corresponding LPS with P. penneri 31 O-antiserum. This ¢nding suggests that the corresponding antibody may recognise a larger epitope, including GlcNAc6PEtn and a neighbouring sugar. Previously, the importance of EtnP and its N-[(R)-1carboxyethyl] derivative (D-AlaEtnP) in manifesting the serological speci¢city has been demonstrated for some other Proteus O-antigens. For instance, the presence of EtnP in the O-antigen of P. mirabilis O17 is one of the factors that are responsible for its serological distinctions from the O-antigen of P. penneri 20 and some other cross-reactive P. penneri strains, which was the basis for classi¢cation of the P. mirabilis and P. penneri strains in one Proteus serogroup O17 as two di¡erent subgroups [24]. On the other hand, two serologically related P. mirabilis strains, PrK 28/57 and EU313, have been classi¢ed into the same Proteus serogroup O14 based on serological data and the presence in their O-antigens of K-D-Galp6(D-AlaEtnP) as the only common component [26].

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

Acknowledgements [15]

This work was supported by the Russian Foundation for Basic Research (grant 02-04-48767), INTAS (grant YS 2001-2/1) and the Sciences Research Committee (KBN, Poland, grant 6 P04A 074 20).

[16]

[17]

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