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Infection and Immunity, October 2004, p. 5925-5930, Vol. 72, No. 10
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.10.5925-5930.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of the Antigenic Lipopolysaccharide O Chain and the Capsular Polysaccharide Produced by Actinobacillus pleuropneumoniae Serotype 13

Leann L. MacLean, Malcolm B. Perry,* and Evguenii Vinogradov

Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada

Received 29 April 2004/ Returned for modification 25 June 2004/ Accepted 1 July 2004


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ABSTRACT
 
Serotyping of Actinobacillus pleuropneumoniae, the etiologic agent of porcine pleuropneumonia, is important for epidemiological studies and for the development of homologous vaccine cell preparations. The serology is based on the specific chemical structures of capsular polysaccharides (CPSs) and lipopolysaccharide (LPS) antigenic O-polysaccharide moieties (O-PSs), and knowledge of these structures is required for a molecular-level understanding of their serological specificities. The structures of A. pleuropneumoniae serotype 1 to 12 CPSs and O-PSs have been elucidated; however, the structures associated with three newly proposed serotypes (serotypes 13, 14, and 15) have not been reported. Herein we described the structures of the antigenic O-PS and CPS of A. pleuropneumoniae serotype 13. The O-PS of the A. pleuropneumoniae serotype 13 LPS is a polymer of branched tetrasaccharide repeating units composed of L-rhamnose, 2-acetamido-2-deoxy-D-galactose, and D-galactose residues (1:1:2). By use of hydrolysis, methylation, and periodate oxidation chemical methods together with the application of one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy and mass spectrometry, the structures of the O chain and CPS were determined. The CPS of A. pleuropneumoniae serotype 13 was characterized as a teichoic-acid type polymer. The LPS O antigen was identical to the O-PS produced by A. pleuropneumoniae serotype 7. The CPS has the unique structure of a 1,3-poly(glycerol phosphate) teichoic acid type I polymer and constitutes the macromolecule defining the A. pleuropneumoniae serotype 13 antigenic specificity.


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INTRODUCTION
 
Actinobacillus pleuropneumoniae is a gram-negative bacterium causing contagious pleuropneumonia in pigs. Based on NAD requirements, A. pleuropneumoniae can be divided into biovar 1 strains, which are NAD dependent, and biovar 2 strains, which are NAD independent. To date, 15 serotypes, based on carbohydrate antigens, have been described (5), and serotypes 1 and 5 have been subdivided into 1a and 1b and 5a and 5b, respectively (17, 23).

The chemical structures of the carbohydrate antigens found in the capsules and the O-polysaccharide components (O-PSs) of the lipopolysaccharides (LPSs) of A. pleuropneumoniae serotypes 1 to 12 (27) and serotype 14 (28) have been elucidated and have assisted in explaining observed serological specificities and cross-reactions. Recently, three new serotypes of A. pleuropneumoniae have been described, serotypes 13 and 14, which are NAD independent strains (biovar 2) (26), and serotype 15 (biovar 1), the predominant serotype present in Australian pigs (5).

Knowledge of the serotypes prevalent in a geographic region is important (4) since current vaccines involving killed whole-cell bacterial preparations can protect only against infection by a homologous serotype present in the vaccine (7, 14, 24, 25, 30). We have undertaken the characterization of the newly described serotypes of A. pleuropneumoniae, and this paper describes the elucidation of the structure of the LPS O-PS and the capsular polysaccharide (CPS) of A. pleuropneumoniae serotype 13.


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MATERIALS AND METHODS
 
A. pleuropneumoniae serotype 13 (NRCC 6230; Fodor strain N-273 [9, 26]), the reference strain from the collection of M. Gottschalk (Laval University), was grown on chocolate agar plates (Oxoid) incubated aerobically at 37°C overnight and used to inoculate 1 liter of medium in 4-liter baffle flasks (medium, brain heart infusion broth [Difico; 37g/liter]) supplemented with hemin (Sigma H-2250) to a final concentration of 5 mg/liter. Stock D-glucose was added to a final concentration of 10% (wt/vol). The 1-liter culture was grown in a New Brunswick Scientific G26 psycrotherm at 37°C and 175 rpm for 6.5 h. Twenty-three liters of medium in an MBR 30-liter fermentor was inoculated with the 6.5-h culture, and growth was continued at 37°C with dissolved oxygen controlled at 20% saturation. At 17 h the culture was killed by the addition of phenol to a 5% concentration and the cells were harvested by using a Cepa Z41 continuous centrifuge (yield, 355 g [wet paste]).

Preparation of LPS, CPS, and O-PS. A cell paste of A. pleuropneumoniae serotype 13 (355 g) was extracted with stirred hot 50% aqueous phenol (550 ml, 70°C, 15 min), and the separated phenol and aqueous phases from the cooled (4°C) extract were dialyzed against tap water until phenol free and then lyophilized. The products were resuspended in 60 ml of 0.02 M sodium acetate (pH 7.0) and were treated sequentially with RNase, DNase, and proteinase K (37°C, 3 h each). Trace solids were removed from the digest by low-speed centrifugation, the samples were subjected to ultracentrifugation (105,000 x g, 4°C, 12 h), and the precipitated gels were dissolved in water and lyophilized to yield 2.22 g (aqueous phase) and 84 mg (phenol phase) of LPS.

The supernatants were treated with cold acetone (6 volumes), and the precipitated products (crude CPS) were collected by centrifugation. The aqueous-phase precipitate (1.90 g) of crude putative CPS upon Sephadex G-50 gel filtration yielded a high-molecular-mass polysaccharide (Kav, 0.03 to 0.04; 320 mg [distribution coefficient Kav = (Ve – V0)/(Vt – V0), where Ve is the elution volume of the specific material, V0 is the void volume of the system, and Vt is the total volume of the system]) with an [{alpha}]D + 99o (c 0.3, water) that was used in subsequent analyses.

O-deacetylated CPS was prepared by treatment of native CPS (30 mg) with 0.02 M ammonium hydroxide (5 ml) at 37°C for 4 h and following dilution with water was lyophilized to yield acetate-free CPS (27 mg) with an [{alpha}]D + 62o (c 1.4, water).

Aqueous-phase LPS (0.5 g) was hydrolyzed with 2% (vol/vol) acetic acid (AcOH) (75 ml, 100°C, 2 h), and following the removal of precipitated lipid A (117 mg) the lyophilized water-soluble products were fractionated by Sephadex G-50 column chromatography to yield a high-molecular-mass O-PS (Kav, 0.03 to 0.04; 224 mg), a core oligosaccharide (Kav, 0.34; 80 mg), and a low-molecular-mass fraction (Kav, 0.94; 40 mg) containing KDO (3-deoxy-D-octulosonic acid).

Chromatography. Gas chromatography was performed using a ZB-50 column (30 m by 0.25 mm; Phenomenex) in an Agilent 6850 chromatograph fitted with a flame ionization detector or a Varian Saturn 2000 ion-trap gas chromatography-mass spectrometry (MS) instrument and a temperature program of 170°C (delay, 2 min) at 2°C/min to 220°C. Retention times and mass spectra were matched with authentic reference samples. Gel filtration chromatography was done with either Sephadex G-50 or Bio-gel P2 columns as previously described (20).

NMR spectrometry. For nuclear magnetic resonance (NMR) spectrometry, 1H, 13C, and 31P spectra were recorded with a Varian 400-MHz spectrometer with samples in D2O and referenced to an internal acetone standard (1H, 2.225 ppm; 13C, 31.07 ppm). COSY, TOCSY, NOESY, HSQC, and HMBC experiments were done as previously described (31).

Periodate oxidation. Smith-type oxidation (11) of O-PS was done as previously described (1).

O deacetylation. CPS (29 mg) dissolved in 0.02 M ammonium hydroxide (5 ml) was kept at 37°C for 4 h, and following dilution with water (10 ml) and lyophilization, the void volume fraction obtained by Sephadex G-50 gel filtration was collected, lyophilized (yield, 27 mg), and used for NMR analysis.

Colorimetric analyses. Determination of O-acetyl was made as described by Hestrin (15), and phosphate was determined by the method of Chen et al. (6).


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RESULTS AND DISCUSSION
 
Extraction of fermentor-grown cells of the reference strain of A. pleuropneumoniae serotype 13 by a modified hot aqueous phenol method (16) released into solution the LPS and CPS produced by the bacterium. The aqueous phase of the cooled extract upon ultracentrifugation yielded a precipitated gel that was dissolved in water and lyophilized to give LPS (6% yield based on dry cell mass). Dilution of the supernatant with acetone yielded a precipitate of crude CPS that upon Sephadex G-50 column gel filtration gave a high-molecular-mass fraction (Kav, 0.02 to 0.03; 3% yield based on dry cell mass) of pure CPS.

Mild acid hydrolysis of the LPS (2% AcOH, 100°C, 2 h) yielded a precipitate of released lipid A (23%), and gel filtration column chromatography of the water-soluble products gave a high-molecular-mass O-PS (45%), a core oligosaccharide fraction (16%), and a low-molecular-mass fraction containing KDO (8%).

The O-PS had an [{alpha}]D + 24o (c 0.3, water) and on acid hydrolysis yielded L-rhamnose, D-galactose, and 2-amino-2-deoxy-D-galactose in the molar ratio 1:2:1. Preparative paper chromatography afforded chromatographically pure samples of each glycose, which were fully characterized from their paper chromatographic mobilities, gas-liquid chromatography (GLC)-MS analysis of their reduced (sodium borodeuteride [NaBD4]) and acetylated alditol-1d derivatives (12), and determination of their configurations by GLC analysis of their acetylated derived 2-(R)-butyl-glycosides (10).

Consideration of the glycose composition and the one-dimensional (1D) 1H and 13C NMR spectra of the O-PS indicate that the O antigen is composed of a repeating tetrasaccharide unit. The proton spectrum (Fig. 1; Table 1) showed four equal-area anomeric proton resonances (4.42 to 5.03 ppm), together with signals at 1.30 (3H) ppm arising from H-6 of the L-Rha constituent and at 2.03 ppm (3H) from the N-acetyl (CH3) substituent of the 2-acetamido-2-deoxy-D-galactose (D-GalNAc) constituent. Consistent with the proton NMR data, the 13C NMR spectrum (Table 1) showed four anomeric carbon signals (102.0 to 105.4 ppm), a signal at 17.5 ppm from the C-6 of the L-Rha constituent, a resonance at 52.6 ppm characteristic for a 2-deoxy-2-acetamido C-2 substitution of a D-GalNAc residue, and inter alia resonances at 22.8 ppm (NH-COCH3) and 175.5 ppm (NH-COCH3), also characteristic of the N-acetyl substitution of the GalNAc O-PS constituent.



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  		FIG. 1. 1H NMR spectrum of the O-PS of A. pleuropneumoniae serotype 13. Indicated are the four anomeric signals (H-1) of the component glycose units: ->4)-{alpha}-L-Rha-(1-> (A), ->3)-ß-D-Gal-(1-> (B), ->3,4)-ß-D-GalNAc-(1-> (C), and ß-D-Gal-1->. Also indicated are the NAc methyl signal of C and the C-6 methyl signal of A.


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  		TABLE 1. NMR spectral data for the LPS O polysaccharide of A. pleuropneumoniae serotype 13

Methylation analysis (13) and GLC-MS identification of the reduced (NaBD4) and acetylated hydrolysis products of the fully methylated polymer identified the acetylated, reduced (NaBD4) alditol derivatives 2,3-di-O-methylrhamnitol-1d, 2,3,4,6-tetra-O-methylgalactitol-1d, 2,4,6-tri-O-methgalactitol-1d, and 2-deoxy-2-(N-methylacetamido)-6-O-methyl-galactitol-1d (1:1:1:1). The analysis is consistent with the presence of the respective linkage units: ->4)-L-Rhap-(1->, D-Galp-(1->, ->3)-D-Galp-(1-> and ->3,4)-D-GalNAc(1->.

2D NMR methods were employed to determine the interglycosidic linkages and the anomeric configurations of the above units within the O-PS. The assignments of chemical shifts of the proton and carbon atoms in the O-PS were established from COSY and TOCSY analysis, and carbon-proton correlations were established from HSQC and HMBC measurements (Fig. 2; Table 1). From the respective coupling constants JH-1,H-2 (<2 Hz) and JC-1,H-1 (171 Hz), the L-Rhap could be assigned the {alpha} configuration, while D-Galp and D-GalpNAc, with corresponding coupling constants of 7.2 to 8.2 Hz and 163 to 165 Hz, respectively, allowed them to be assigned the ß configuration. A 2D phase-sensitive NOESY experiment provided interresidue data consistent with the methylation linkage analysis, thus establishing the structure of the O-PS as:

{zii01004429500e1}



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  		FIG. 2. HSQC NMR spectrum of the O-PS of A. pleuropneumoniae serotype 13 showing proton and carbon correlation cross peaks for the component glycose units: ->4)-{alpha}-L-Rha-(1-> (A), ->3)-ß-D-Gal-(1-> (B), ->3,4-ß-D-GalNAc-(1-> (C), and ß-D-Gal-(1-> (D).

Further proof of the above structural assignment was provided by a Smith-type periodate oxidation (11) in which Rhap residue A and D-Galp residue D were oxidized. Following reduction (NaBH4) and mild hydrolysis of the oxidized polymer, a trisaccharide was identified by chemical and NMR analysis to have structure II, the expected degradation product from the proposed O-PS structure I:

{zii01004429500e2}

The accumulated experimental data provide convincing evidence that the structure of the LPS O-PS of A. pleuropneumoniae serotype 13 is identical to that previously determined for the O antigen of A. pleuropneumoniae serotype 7 (3). The O-PS structure is similar to that of the O-PS of A. pleuropneumoniae serotype 4 (2), differing only in the replacement of the D-GalpNAc by a 1,3-linked ß-D-Glcp side-group residue instead of a 1,3-linked ß-D-Galp side-group residue. It is interesting that Lebrun et al. (19) showed that monoclonal antibodies can be specific or nonspecific for the respective O-PSs of A. pleuropneumoniae serotypes 4 and 7, a finding consistent with the chemical structures, which suggest that the O-PSs should share similar carbohydrate epitopes and yet should also have unique structural features involving single nonreducing ß-D-Galp or ß-D-Glcp side end-group residues.

In order to prevent serological misidentification of A. pleuropneumoniae serotype 13 due to cross-reacting antibody to common LPS O antigen, specific type 13 antibody may be made by using A. pleuropneumoniae serotype 13 CPS or its conjugate as an immunogen. Alternatively, polyclonal antisera prepared against whole A. pleuropneumoniae serotype 13 cells may be adsorbed out by A. pleuropneumoniae serotype 7 cells or by insolublized LPS (8) from either A. pleuropneumoniae serotype 7 or 13.

The water phase of the phenol-extracted A. pleuropneumoniae serotype 13 cells, after removal of LPS by ultracentrifugation and following precipitation with acetone, yielded a high-molecular-mass product which upon purification by Sephadex G-50 column chromatography gave a fraction (Kav, 0.01 to 0.05) which had an [{alpha}]D +99o (c 0.3, water) and on hydrolysis yielded D-galactose and glycerol (1:1), identified by GLC-MS of derived glycitol acetates, the D-Gal being further characterized by GLC of its acetylated 2-(R)-butyl glycoside derivatives. Colorimetric phosphate analysis (6) showed the product to contain 7.1% phosphorus, leading to the conclusion that the capsular material was composed of equimolecular amounts of D-galactose, glycerol, and phosphate. Colorimetric analysis (15) indicated that the native CPS contains a total 1.0 M O-acetyl substitution of the D-Galp residue.

Mild basic hydrolysis or dephosphorylation of the CPS with cold 48% aqueous HF resulted in depolymerization products and the quantitative chromatographic isolation of compound III, composed of D-galactose and glycerol (1:1), and it had an [{alpha}]D + 158o (c 0.2, water). The 1H and 13C NMR spectra of compound III showed chemical shifts (Fig. 3; Table 2) that identified it as {alpha}-D-galactopyranosyl-(1->2)-glycerol. The spectra showed all the expected signals for an {alpha}-D-Galp linked to the O-2 of a glycerol residue, following from the observation of an NOE correlation between Gal H-1 and glycerol (Gro) H-2. The glycerol being phosphorylated at O-1 and O-3 led to low-field shifts of H-1, H-3, C-1, and C-3, as seen in comparing 1H and 13C to their shifts in O-deacetylated CPS (Table 2). The occurrence of the anomeric carbon signal of the D-Galp residue seen at 97.5 ppm with J C-1,H-1 174 Hz with the corresponding proton signal at 5.15 ppm with a coupling constant JH-1,H-2 3.8 Hz, considered in conjunction with the high positive specific optical rotation of compound III, confirms the {alpha} configuration of the D-Galp moiety and the proposed structure. The characterization of compound III indicates that the CPS is a polymer of a basic backbone chain of C-1 and C-3 phosphate ester-linked glycerol residues bearing single {alpha}-D-Galp residues glycosidically linked at the C-2 positions of each glycerol unit.



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  		FIG. 3. HSQC NMR spectrum of compound III from A. pleuropneumoniae serotype 13 CPS showing carbon and proton correlation cross peaks for the {alpha}-D-galactose (Gal) and glycerol (Gro) components.


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  		TABLE 2. NMR spectral data for compound III from the CPS and the O-deacetylated CPS of A. pleuropneumoniae serotype 13

2D NMR studies of the polymeric O-deacetylated CPS (Table 2) confirmed the above proposed basic CPS structure. Further analysis of the NMR spectra of the native CPS revealed that the O-3 and O-4 positions of the D-Galp residues were acetylated to the extent of 40 and 60%, respectively. The positions of the O-acetyl substituents were inferred from a consideration of the chemical shifts in the NMR carbon and proton resonances about the {alpha}-D-Gal O-3 and O-4 positions compared with the chemical shifts observed for the same proton and carbon atoms in the O-deacetylated CPS. While it is known that the presence of O-acetyl groups can determine antigenic specificities (18), it is also known that the degree and position of O acetylation are often dependent on cell culture conditions, and batch-to-batch production changes in O-acetyl position and degree of substitution are often found. In the present study some batch CPS preparations showed a minor (<4%) O-acetyl substitution at the C-2 Galp position. The combined results of the chemical and physical analyses of the currently studied A. pleuropneumoniae serotype 13 CPS show the polymer to have structure IV.

{zii01004429500e4}

The structural occurrence of 1,3 poly(glycerol phosphates) (type I teichoic acids) is not unusual. They have been found in many bacterial species (21, 22, 29), and glycosyl substitution at C-2 by the monosaccharide units of glucose, galactose, rhamnose, and 2-acetamido-2-deoxyhexose have been demonstrated.


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ACKNOWLEDGMENTS
 
We thank M. Gottschalk for strains of A. pleuropneumoniae and Perry Flemming for the fermentor production of bacterial cell mass.

We thank the Canadian Bacterial Diseases Network, Center of Excellence, for their support.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Biological Sciences, National Research Council, 100, Sussex Dr., Ottawa, Canada K1A 0R6. Phone: (613) 990-0837. Fax: (613) 941-1327. E-mail: malcolm.perry{at}nrc-cnrc.gc.ca. Back

Editor: D. L. Burns


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Infection and Immunity, October 2004, p. 5925-5930, Vol. 72, No. 10
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.10.5925-5930.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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