Purification and Characterization of an Exopolysaccharide of ...

INFECTION AND IMMUNITY, Oct. 1995, p. 3959–3965 0019-9567/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 63, No. 10

Purification and Characterization of an Exopolysaccharide of Burkholderia (Pseudomonas) pseudomallei I. STEINMETZ,1* M. ROHDE,2

AND

B. BRENNEKE1

Institute of Medical Microbiology, Hannover Medical School, 30625 Hannover,1 and Gesellschaft fu ¨r Biotechnologische Forschung mbH, 38124 Braunschweig,2 Germany Received 17 April 1995/Returned for modification 20 June 1995/Accepted 12 July 1995

Burkholderia pseudomallei (basonym Pseudomonas pseudomallei) is the causative organism of melioidosis, a disease which is recognized as a major public health problem primarily in Southeast Asia and Northern Australia. In this paper, we report on the identification, purification, and characterization of a species-specific exopolysaccharide of B. pseudomallei. After immunization of mice with a B. pseudomallei strain exhibiting mucoid growth characteristics, we isolated an immunoglobulin G1 monoclonal antibody (MAb) (3015) with specificity for a carbohydrate structure as determined by immunoblotting following sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electron microscopy studies with MAb 3015 revealed reactivity with an exopolysaccharide with a capsule-like appearance in the immunizing strain. All of the mucoid and nonmucoid B. pseudomallei strains tested from geographically different tropical regions were recognized by MAb 3015 in an enzyme-linked immunosorbent assay or immunoblot, indicating that the exopolysaccharide is constitutively expressed among this species. Intensive testing for cross-reactivity including members of all the Pseudomonas rRNA groups showed no cross-reactivity except in the case of the closely related species Burkholderia mallei. A protocol for purification of the exopolysaccharide which is based principally on mechanical separation from the cell surface followed by repetitive ethanol precipitation steps and finally affinity chromatography using MAb 3015 was established. The exopolysaccharide yielded was of high purity. Gel permeation chromatography was performed, and the molecular mass was estimated to be >150 kDa. Sera from patients with melioidosis were strongly reactive with the purified exopolysaccharide, indicating its in vivo expression and immunogenicity in natural infection. The diagnostic value of the exopolysaccharide and its role in the pathogenesis of disease must still be determined. nase, lipase, and dermonecrotic protease), and a heat-labile exotoxin (11, 20). Exopolysaccharides were among the first known bacterial virulence determinants in gram-negative and gram-positive bacteria. They include structures from discrete capsules to massive slime wall formations outside the outer membrane of the bacterial cell. During this study, we developed a monoclonal antibody (MAb) with specificity for an exopolysaccharide of B. pseudomallei which serves as a tool for purification of the exopolysaccharide and for investigating its characteristics and distribution among this species. We report the existence of a constitutively expressed exopolysaccharide among B. pseudomallei strains that has not previously been described.

Burkholderia pseudomallei, previously known as Pseudomonas pseudomallei (25), is the causative organism of melioidosis, an infectious disease of humans and animals which is endemic primarily in Southeast Asia and Northern Australia. The gramnegative bacillus is a saprophyte, ubiquitous in the soil and surface water of areas of endemicity (14, 22). Infection is thought to occur by ingestion, inhalation, or inoculation of the environmental organisms (14, 22). In Thailand, the rice farming population is at particular risk of acquiring the infection, and it is assumed that in the majority of cases infection occurs because of soil contamination of minor cuts and abrasions (2, 7). It seems likely that melioidosis is greatly underdiagnosed, especially in poor, rural areas where appropriate microbiological facilities are not available, and the true worldwide importance remains unknown (8). The clinical manifestation ranges from subclinical to acute localized forms, acute septicemic, and chronic forms (6, 14). Although apparently healthy individuals can be infected, septicemic melioidosis is significantly associated with underlying diseases, most commonly diabetes mellitus or chronic renal failure (2). It is now known to be a major cause of morbidity and mortality in certain areas of the tropics (8). In contrast to the increasing knowledge of the epidemiology of the disease, the pathogenesis of melioidosis is still poorly understood. Reports of long periods of latency and relapses of melioidosis are suggestive of an intracellular survival of B. pseudomallei (6). Putative virulence factors include lipopolysaccharide (LPS), extracellular enzymes (e.g., lecithi-

MATERIALS AND METHODS Bacterial strains and cultivation. All of the bacterial strains used in this study are listed below (see Tables 1 and 2). LMG strains included type strains from the Belgian Collection of Culture Strains (Universiteit Gent, Laboratorium voor Microbiologie, Gent, Belgium), and ATCC and NCTC strains were from the American Type Culture Collection or the National Collection of Type Cultures, respectively, bacteria were grown at 378C on sheep blood agar unless otherwise stated. Production of MAbs. Six-week-old female BALB/c mice (Zentralinstitut fu ¨r Versuchstierkunde, Hannover, Germany) were immunized intraperitoneally once a week for 4 weeks with 2 3 108 heat-inactivated (808C for 1 h in buffer A [0.01 M potassium phosphate buffer made isotonic with saline; pH 7.5]) B. pseudomallei NCTC 7431 cells. The first injection was given with complete Freund’s adjuvant. The second injection was given with incomplete Freund’s adjuvant (Difco). The following injections were given with physiological saline. At the end of the immunization regimen, the mice were splenectomized and the spleen cells were fused with X63-Ag8.653 myeloma cells as described previously (18). The culture supernatant fluids of growing clones were screened by enzymelinked immunosorbent assay (ELISA) with B. pseudomallei NCTC 7431 whole cells as an antigen (see below). The resulting hybridomas were cloned by limiting

* Corresponding author. Mailing address: Medical Microbiology, Hannover Medical School, Konstanty-Gutschow-Str. 8, 30625 Hannover, Germany. Phone: 0511-532-4340. Fax: 0511-532-4366. 3959

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dilution. For the production of ascites fluid, hybridoma cells were injected intraperitoneally into pristane-pretreated BALB/c mice. ELISA methods. The ELISA for the detection of MAbs against B. pseudomallei was performed in U-shaped wells of nonflexible polystyrol microtiter plates (Greiner, Nu ¨rtingen, Germany). The plates were coated with heat-treated B. pseudomallei NCTC 7431 cells grown on sheep blood agar plates as follows. The cells were washed with physiological saline and adjusted in buffer A to 2 3 108 cells per ml. A 20-ml volume of the suspension was added to each well, and the plates were left for 2 h at room temperature. The plates were washed twice with 200 ml of buffer A and then incubated with buffer A-BSA (buffer A containing 1% [wt/vol] bovine serum albumin; Merck, Darmstadt, Germany) for 30 min. Hybridoma culture supernatant fluids (20 ml) and culture medium as a negative control were then added, and the plates were incubated for 2 h and then washed extensively with buffer A prior to addition of 10 ml of biotin-labeled goat-antimouse immunoglobulin (Jackson Immunoresearch Laboratories) (diluted 1:2,000 in buffer A-BSA) and incubation for 30 min. The plates were then washed with buffer A, 10 ml of streptavidin coupled to b-galactosidase (Boehringer GmbH, Mannheim, Germany) and diluted 1:2,000 in buffer A-BSA was added, and the plates were incubated for 30 min. The microtiter plates were then washed twice with buffer A and once with 0.01 M sodium phosphate buffer, pH 6.9. Substrate solution (20 ml of 0.1 mM 4-methylumbelliferyl-b-D-galactopyranoside [Boehringer] in 0.01 M sodium phosphate buffer, pH 6.9) was added to each well, and the fluorescent product was measured in a Titertek Fluoroskan 2 (Labsystems, Helsinki, Finland) as relative fluorescence units, with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. When single fractions were analyzed after affinity or gel chromatography for the presence of B. pseudomallei exopolysaccharide, the same ELISA was used but with two modifications. The plates were coated with 20 ml of each fraction as the antigen, and MAb 3015 supernatant was used as the detecting antibody. Testing of human serum specimens from melioidosis patients (four patients who acquired infection in Thailand and one patient in Malaysia) for reactivity with the exopolysaccharide was performed with the same test principle but using a different developing system. The plates were coated with 20 ml of affinity-purified exopolysaccharide (50 mg/ml, diluted in buffer A) for 2 h. After incubation of sera (20 ml, diluted in buffer A-BSA) for 2 h, 10 ml of peroxidase-labeled goat anti-human immunoglobulin (Dianova, Hamburg, Germany) (diluted 1:3,000 in buffer A-BSA) was added and the solution was incubated for 30 min. As a substrate solution, 20 ml of 2.7% 2,29-azino[di(3-ethylbenzthiazolinesulfonic acid)] (Sigma, Delsenhofer, Germany) in potassium phosphate buffer (pH 6.0) containing 0.0025% H2O2 was used, and the A414 was measured in a Titertek Multiscan (Flow Laboratories, Meckenheim, Germany). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. SDS slab gel electrophoresis was performed as described previously (18). Bacteria grown on agar plates were washed twice with physiological saline and adjusted to a concentration of 5 3 109 cells per ml. After centrifugation in an Eppendorf Biofuge at 10,000 3 g for 5 min, the pellet from 1 ml of the bacterial suspension was suspended in 1 ml of sample buffer (0.0625 M Tris-hydrochloride [pH 6.8], 5% 2-mercaptoethanol, 2% SDS, 12.5% glycerol) and heated for 10 min at 1008C. A sample of 20 ml (equivalent to 108 bacteria) was applied to each lane of the gel. Molecular weights were estimated by comparison with a prestained SDS marker kit (MW-SDS-Blue; Sigma). When purified exopolysaccharide or bacterial extracts were examined, 8 mg was applied to each lane of the gel. The blotting procedure was done by previously described methods (18) with the minor modification of using 200 V for 3 h. The nitrocellulose sheet was saturated with 2% (wt/vol) instant dried milk in buffer A overnight. MAb 3015 was purified by affinity chromatography on protein ASepharose (Pharmacia, Uppsala, Sweden) and diluted 1:2,000 in 2% instant dried milk-buffer A. Peroxidase-conjugated goat anti-mouse immunoglobulin (Jackson Immunoresearch Laboratories) (diluted 1:2,000 in 2% dried milkbuffer A) was used for final development. The substrate was 4-chloro-1-naphthol (Sigma). Purification of B. pseudomallei exopolysaccharide. Mucoid strain NCTC 7431 was grown to confluence on modified Ashdown agar plates (5) for 72 h at 378C. Colonies were harvested with 20 ml of 0.01 M phosphate-buffered saline (PBS) (0.01 M sodium phosphate buffer made isotonic with saline; pH 7.4) per plate. The collected mucoid material was pooled and stirred vigorously with a magnetic stirrer for 1 h to separate the mucoid layer from the cells and to obtain a uniform solution. The solution was then centrifuged for 4 h at 20,000 3 g at 48C to remove the whole bacteria. The supernatant was heated for 30 min at 808C to kill the remaining viable bacteria and centrifuged for 30 min at 20,000 3 g at 48C. The supernatant was precipitated by adding cold absolute ethanol to a final concentration of 80% (vol/vol) for 1 to 2 h at 2208C. The precipitate was collected by centrifugation for 30 min at 3,000 3 g at 48C, washed once in 80% ethanol for 30 min, and again centrifuged for 30 min. This washing step was repeated once with 96% ethanol. The precipitate was then dissolved in 0.01 M PBS (containing 10 mM MgCl2 and 1 mM CaCl2), and RNase A (100 mg/ml; Sigma) and DNase I (100 mg/ml; Sigma) were added and the mixture was incubated for 2.5 h at 378C to remove the contaminating RNA and DNA. After incubation, enzymes were inactivated and denatured by being heated for 30 min at 808C and then subjected to centrifugation for 30 min at 20,000 3 g at 48C. The supernatant was used for a second precipitation procedure with 80% ethanol as described above, and after centrifugation, the precipitate was dissolved in 2 ml of deionized water. This

INFECT. IMMUN. crude material was then used for affinity chromatography. Immunoglobulin G1 (IgG1) MAb 3015 was coupled to protein A-Sepharose beads (Pharmacia) by using dimethylpimelimidate as a coupling reagent as described elsewhere (10). The column was loaded with 1 mg of the crude material per 2.5 ml of wet beads. For elution of the polysaccharide from the column, 100 mM triethylamine (pH 11.5) was used. Fractions were collected and immediately neutralized with acetic acid. Each fraction was tested in the ELISA (see above) for the presence of exopolysaccharide by using MAb 3015, and positive fractions were pooled, dialyzed against aqua dest overnight, and finally lyophilized. The purity of the exopolysaccharide preparation was analyzed by SDS-PAGE followed by a highly sensitive silver staining procedure for the detection of proteins (23) and LPS (24). For a more sensitive detection of LPS, a quantitative Limulus amoebocyte lysate assay was kindly performed by Behringwerke AG, Marburg, Germany. The A260 was measured for DNA and RNA detection. Preparation of LPS-containing bacterial extracts. B. pseudomallei NCTC 7431 cells were harvested, killed with 0.5% phenol, and then washed once with ethanol, twice with acetone, and once with diethyl ether. The bacteria were dried and then digested with RNase A (100 mg/ml), DNase I (100 mg/ml), and proteinase K (100 mg/ml) (all from Sigma). After extensive dialysis, the extract was lyophilized. Gel permeation chromatography. Gel filtration for molecular mass determination was performed on a column (1.6 by 50 cm) of Sepharose CL-4B (Pharmacia). The column was equilibrated in 0.01 M PBS (pH 7.4), and polysaccharides were eluted with the same buffer at a flow rate of 24 ml/h. Fractions of 1.8 ml were collected. The void volume and total volume were determined with blue dextran 2000 (Pharmacia) and acetone, respectively. Dextrans of 670 and 150 kDa (Fluka Biochemica, Neu-Ulm, Germany) were used as standards and detected by phenol-sulfuric acid assay (10). A 20-mg amount of purified polysaccharide dissolved in 500 ml of PBS was applied to the column. Fractions were tested for the presence of the polysaccharide by ELISA as described above. Postembedding labeling and immunoelectron microscopy. For the preservation of capsular structures, the bacteria were pretreated (i) with 1:50-diluted ascites fluid with the specific IgG1 MAb 3015 for 45 min at room temperature, (ii) with IgG1 MAb 2454 specific for the Haemophilus influenzae type B polysaccharide for 45 min at room temperature as a control, and (iii) without any pretreatment. After three washing steps with 0.1 M PBS (0.1 M sodium phosphate buffer, 0.15 M NaCl; pH 6.9), the bacteria were fixed with 0.5% formaldehyde and 0.2% glutaraldehyde (final concentrations) in 0.1 M PBS for 1 h on ice. After three washes with 0.1 M PBS containing 10 mM glycine to block free aldehyde groups, the cells were embedded by progressively lowering the temperature with Lowicryl K4M resin (21). The following modifications of the method were made: (i) after dehydration in 10% ethanol, the samples were treated with 0.5% uranyl acetate in 10% ethanol for 1 h on ice; (ii) the infiltration step with 1 part ethanol and 1 part K4M resin was performed overnight; (iii) the infiltration step with 1 part ethanol and 2 parts K4M resin lasted for 12 h; and (iv) infiltration with pure K4M resin lasted for 2 days. After polymerization of the samples for 2 days at 2358C, samples were trimmed and polymerized for another day at room temperature. Ultrathin sections were incubated with MAb 3015 or the control MAb (200 mg of IgG1 per ml) overnight at 48C and washed with 0.1 M PBS, and then the sections were incubated with protein A-gold complexes (10-nm diameter; concentration giving an A520 of 0.02). The sections were subsequently rinsed with 0.1 M PBS containing 0.01% Tween 20 and then with distilled water. After being air dried, the sections were counterstained with 4% aqueous uranyl acetate (pH 4.5) for 5 min. Samples were examined with a Zeiss EM 910 electron microscope at an acceleration voltage of 80 kV at calibrated magnifications.

RESULTS Reactivity of MAb 3015, specific for a B. pseudomallei exopolysaccharide, in immunoblot and electron microscopy. In order to generate MAbs with specificity for an exopolysaccharide, mice were immunized with heat-inactivated whole cells of B. pseudomallei NCTC 7431, which has mucoid growth characteristics. Screening of hybridoma supernatants in an ELISA with the same antigen that was used for immunization led to the isolation of several MAbs which react strongly with B. pseudomallei NCTC 7431 whole cells. IgG1 MAb 3015 was selected from these clones for further studies. The antibody was tested against whole-cell lysates in an immunoblot following SDS-PAGE and revealed reactivity with a diffuse band in a high-molecular-mass range (Fig. 1). The reactivity of MAb 3015 could not be eliminated by incubating the immunoblot with proteinase K. These data were suggestive of a surfacelocated carbohydrate epitope recognized by MAb 3015. We hypothesized that the antibody was reactive with the mucoid substance produced by the immunizing strain, and conse-

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BURKHOLDERIA PSEUDOMALLEI EXOPOLYSACCHARIDE

FIG. 1. Immunoblot with MAb 3015 of SDS-polyacrylamide (3 to 10% polyacrylamide) gel with affinity-purified exopolysaccharide and whole bacterial cells of B. pseudomallei NCTC 7431. Lanes: 1, exopolysaccharide; 2, whole cells; 3, Pseudomonas aeruginosa 492 alginate as a negative control.

quently immunoelectron microscopy experiments were performed. The results with different experimental conditions are shown in Fig. 2. Figure 2B and C demonstrate reactivity of MAb 3015 with an exopolysaccharide with a capsule-like appearance in the immunizing strain. These results were obtained by incubating the cells with MAb 3015 before fixation in order to cross-link and to preserve the capsule structure. The cells in Fig. 2A were not pretreated in this way. Although cell surface-bound antibody could be detected, the capsule structure was much less preserved. This is probably due to the collapse of the structure during the fixation procedure. Constitutive expression of the exopolysaccharide among B. pseudomallei strains. To investigate the distribution of the exopolysaccharide within the B. pseudomallei species, we tested 12 different strains from different geographical regions, most of them showing a rough-type growth pattern with MAb 3015. All strains were positive in the ELISA and the immunoblot, demonstrating constitutive expression of the exopolysaccharide independent of the growth type and geographical origin (Table 1). The patterns of reactivity of the different B. pseudomallei strains in the immunoblot all appeared the same as that in Fig. 1. We also investigated whether the expression of the exopolysaccharide is dependent on the growth temperature. Different B. pseudomallei strains were grown at 158C, and subsequent testing by immunoblotting and ELISA revealed the same pattern of reactivity with MAb 3015 as for bacteria grown at 378C (data not shown). In order to determine the specificity of MAb 3015, extensive testing for cross-reactivity by ELISA and immunoblotting was performed. Strains from all Pseudomonas rRNA groups were tested together with strains of the former rRNA group II which have now been transferred to the novel genus Burkholderia (25). No cross-reactions could be detected with the exception of the closely related Burkholderia mallei (Table 2). Purification of the exopolysaccharide. B. pseudomallei NCTC 7431, used for immunization, was also used to establish a purification protocol for the exopolysaccharide. This strain produced large amounts of a mucoid substance on modified

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Ashdown agar, so the purification procedure was commenced with bacteria grown under these conditions. The first step involved the mechanical separation of the mucoid substance from the cells by vigorous stirring of a cell suspension and subsequent centrifugation to remove bacterial cells. The resulting supernatant was heat inactivated, and after centrifugation, the supernatant was subjected to a cold 80% ethanol precipitation. The dissolved precipitate was treated with nucleases to remove nucleic acids. After heat inactivation of the enzymes and centrifugation, the supernatant was used for a second ethanol precipitation. During the final purification step, the dissolved precipitate was used for affinity chromatography with MAb 3015, which was bound to protein A-Sepharose. Elution of the bound exopolysaccharide was performed under alkaline conditions. After affinity chromatography, an exopolysaccharide of high purity which gave an orange-yellow color in the phenol-sulfuric acid assay was obtained. The preparation did give a strong signal in the immunoblot using MAb 3015 (Fig. 1). The reaction of the purified preparation was the same as that of wholecell lysates, and this shows that no marked degradation occurred during the purification process (Fig. 1). With highly sensitive silver staining, neither LPS (Fig. 3, lane 1) nor protein (not shown) contamination could be detected. As a control, silver staining of LPS of bacterial extracts of B. pseudomallei after proteinase K digestion was performed, and the results revealed that the LPS had a much lower molecular mass (Fig. 3, lane 2) than the exopolysaccharide detected by immunoblotting of the same preparation, which hardly entered the 15% running gel (Fig. 3, lane 3). In a further analysis, LPS contamination of the affinitypurified exopolysaccharide was calculated to be 0.0025% by the Limulus test. Nucleic acids were not detectable by spectroscopy. Determination of the molecular mass of the exopolysaccharide. The molecular mass of the purified exopolysaccharide was determined on a column of Sepharose CL-4B, which is designed to separate large molecules by gel filtration. The single fractions were tested for the presence of the exopolysaccharide in an ELISA using MAb 3015. First, the elution was performed with PBS containing EDTA, and the exopolysaccharide was detected in a single peak after the void volume (Fig. 4). To obtain an elution pattern under conditions which exclude possible aggregation or micelle formation, the exopolysaccharide was then eluted in the presence of deoxycholate. Figure 4 shows that under those conditions, the peak observed without deoxycholate was completely lost and the main peak of the exopolysaccharide was found at a molecular mass of .150 kDa in comparison with dextran standards. These results suggest that the exopolysaccharide is able to form aggregates which can be dissociated by an anionic detergent. Reactivity of patient sera with the affinity-purified exopolysaccharide. In order to analyze whether the identified exopolysaccharide is immunogenic in humans, sera from patients with septicemic melioidosis were tested against the affinity-purified exopolysaccharide in an ELISA. The strong reactivity of the tested sera (Fig. 5) demonstrates the production of antibodies against this structure in humans and also indicates that the exopolysaccharide is expressed in vivo. In the immunoblot, the reactivity pattern was the same as the pattern found with MAb 3015 (not shown). DISCUSSION There is much evidence for gram-negative and gram-positive bacteria that capsules are important virulence factors of great

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INFECT. IMMUN.

FIG. 2. Immunoelectron microscopic localization of an exopolysaccharide of B. pseudomallei NCTC 7431 using postembedding labeling with MAb 3015. (A) Bacterial cells without pretreatment with the specific MAb 3015 exhibit some labeling on the cell periphery, i.e., the outer membrane region. (B and C) Bacteria preincubated with MAb 3015 show preservation of the capsular structures (arrows). The intensity of the label is higher than that of nontreated cells (A). The exopolysaccharide is located mostly in the capsular structures and not in the outer membrane region of the cell. (D) Sections of bacteria pretreated with MAb 3015 were incubated with a control IgG1 MAb (2454) and then with protein A-gold complexes. Only a very few gold particles (arrowheads) are detectable. The large granules within the cytoplasma most likely represent poly-b-hydroxybutyrate accumulation, which is characteristic for Burkholderia species (17). G, gold particle; OM, outer membrane. Bars, 100 nm (A to C) and 0.5 mm (D).

significance in the pathogenesis of invasive infectious diseases (19). Polysaccharide capsules protect bacteria in their interaction with the host defense systems, e.g., from complement activation and phagocyte-mediated killing (19). B. pseudomallei strains exhibit considerable variations in col-

ony morphology, ranging from the most common rough type to the smooth type and sometimes even to mucoid growth characteristics (5, 16). In this study, immunization with a B. pseudomallei strain showing mucoid growth characteristics led to the isolation of an IgG1 MAb with specificity for a polysac-


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TABLE 1. Reactivity of MAb 3015 with B. pseudomallei strains from different geographical regions B. pseudomallei strain

NCTC NCTC NCTC NCTC NCTC NCTC NCTC NCTC NCTC NCTC NCTC NCTC

4845 1688 4846 6700 7383 7431 8016 10274 10276 8707 8708 11642

Reactivity in:

TABLE 2. Reactivity of MAb 3015 with representative strains of the different Pseudomonas rRNA groups and nonPseudomonas species

Origin

Singapore Malaysia Singapore Malaysia Burma Unknown Australia Malaysia Bangladesh Singapore Singapore Unknown

Immunoblot

ELISA

1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1

charide structure on the bacterial surface. The exopolysaccharide nature of the recognized structure could be determined by using MAb 3015 in immuno-electron microscopy. Bacterial exopolysaccharides can be overlooked by conventional electron microscopy because the dehydration required for this method might completely condense the hydrated polysaccharide matrices, which are then no longer visualized (3). We therefore used preincubation of the cells with the specific MAb in order to cross-link the exopolysaccharide before the fixation procedure, which stabilized the capsule-like structure. This observation is in accordance with the results of previous studies in which specific antibodies were successfully used in similar protocols to demonstrate bacterial glycocalyces (13, 15). MAb 3015 was used to investigate the distribution of the exopolysaccharide among strains of B. pseudomallei. All of the strains were reactive with MAb 3015 in the ELISA with whole cells and in the immunoblot. The strains originate from different tropical regions, and their growth phenotypes range from rough to smooth and include mucoid growth. We therefore conclude that the expression of this exopolysaccharide is a stable characteristic of this species and is not linked to phenotypic growth. Extensive testing for cross-reactivity of MAb 3015 with species from all Pseudomonas rRNA homology groups and nonpseudomonads revealed reactivity with only the closely related B. mallei, which has also been transferred from Pseudomonas rRNA group II to the novel genus Burkholderia (25). This finding is not surprising in view of the DNA and RNA homology of these two species. Furthermore, an antigenic relationship between B. pseudomallei and B. mallei has been found before by using polyclonal antisera (4). Our demonstration of a common exopolysaccharide epitope shared by only these two species further confirms their close relationship. As a basis for further investigations into the biological role of this polysaccharide and for possible diagnostic purposes, a protocol has been developed for purification. By using affinity chromatography as the final step according to this protocol, an exopolysaccharide of high purity was obtained. The exopolysaccharide yielded is likely to be the native structure, since after the mild purification procedure, no signs of breakdown could be observed and immunological detection was still possible. In this preparation, no proteins and nucleic acids were detectable. A very low level of LPS contamination (0.0025%) was measured by Limulus amoebocyte lysate assay. Although determination of endotoxin-like activity by the Limulus amoebocyte lysate assay is very sensitive, it is not entirely specific for LPS. Other polysaccharides may give rise to weakly positive reactions in this assay (1). We do not know the extent to which

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Reactivity in: Strain Immunoblot

ELISA

Pseudomonas rRNA group I Pseudomonas aeruginosa LMG 1242T Pseudomonas alcaligenes LMG 1224 t1 Pseudomonas aureofaciens LMG 1245T Pseudomonas chlororaphis LMG 5004T Pseudomonas cichorii LMG 2162T Pseudomonas fluorescens bv. 1 LMG 1794T Pseudomonas fluorescens bv. 3 LMG 1244 Pseudomonas fluorescens bv. 4 LMG 5939 Pseudomonas mendocina LMG 1223T Pseudomonas pseudoalcaligenes LMG 1225T Pseudomonas putida bv. A LMG 2257T Pseudomonas putida bv. B LMG 1246 t1 LMG 5837 Pseudomonas stutzeri LMG 2243

2 2 2 2 2 2 2 2 2 2 2 2 2 2

2 2 2 2 2 2 2 2 2 2 2 2 2 2

Burkholderia species (former members of rRNA group II) Burkholderia mallei NCTC 10260 NCTC 10230 Burkholderia cepacia LMG 1222T Burkholderia solanacearum LMG 2299T Burkholderia pickettii LMG 5942T LMG 7005

1 1 2 2 2 2

1 1 2 2 2 2

Pseudomonas rRNA group III Acidovorax delafieldii LMG 5943T Acidovorax facilis LMG 2193T Comamonas acidovorans LMG 1226T Comamonas terrigena group 1 LMG 1253T Comamonas testosteroni LMG 1800T Hydrogenophaga flava LMG 2185T Hydrogenophaga palleronii LMG 2366T t1 Hydrogenophaga pseudoflava LMG 5945T

2 2 2 2 2 2 2 2

2 2 2 2 2 2 2 2

Pseudomonas rRNA group IV Pseudomonas diminuta LMG 2089T Pseudomonas vesicularis LMG 2350T

2 2

2 2

Pseudomonas rRNA group V Stenotrophomonas maltophilia ATCC 17448 ATCC 17406

2 2

2 2

Other species Escherichia coli ATCC 11229 ATCC 25922 ATCC 29079 Klebsiella pneumoniae ATCC 10031 NCTC 10995 NCTC 11228

2 2 2 2 2 2

2 2 2 2 2 2

this applies to the B. pseudomallei exopolysaccharide, and, therefore, the level of LPS contamination might be even lower. The purification protocol established in this study constitutes a basis for further investigations into the chemical nature of the exopolysaccharide. We are currently analyzing its monosaccharide composition. The strong reactivity of sera from patients with melioidosis (from Thailand and Malaysia) with the affinity-purified exopolysaccharide of strain B. pseudomallei NCTC 7431 provides additional evidence for the constitutive expression of this structure.

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FIG. 3. Silver staining of SDS–15% polyacrylamide gel with affinity-purified exopolysaccharide of B. pseudomallei NCTC 7431 and bacterial extracts of the same strain after proteinase K digestion for the detection of LPS. Lanes: 1, exopolysaccharide; 2, bacterial extract; 3, immunoblot with MAb 3015 with the same sample and SDS-PAGE gel as in lane 2.

Exopolysaccharides of gram-negative bacteria have been divided into two groups (groups I and II) on the basis of their different structural features and modes of expression (12). The high molecular mass of the B. pseudomallei exopolysaccharide (.150 kDa) estimated by gel filtration and its expression below 208C are both features which suggest that it is a group I exopolysaccharide.

INFECT. IMMUN.

FIG. 5. Reactivities of serum specimens (1:2,500 dilution) from five melioidosis patients and five control serum samples with the affinity-purified exopolysaccharide in an ELISA. Each point is the mean of duplicate determinations.

The identification and characterization of an immunogenic exopolysaccharide highly specific for B. pseudomallei should stimulate investigations in various directions. (i) The applicability of the purified exopolysaccharide as an antigen in a screening test for the detection of antibodies needs to be examined, since currently available tests lack specificity and sensitivity (8). (ii) The exclusive expression of this cell surface structure makes it a potential candidate for antigen detection tests for the rapid diagnosis of melioidosis. (iii) Future work

FIG. 4. Gel filtration chromatography of the affinity-purified exopolysaccharide of B. pseudomallei NCTC 7431 on a column (1.6 by 50 cm) of Sepharose CL-4B. The elution profile as determined by ELISA is shown. F, elution profile with an eluant consisting of 0.1 M PBS (pH 7.4) containing 0.1 M EDTA; E, elution profile with the exopolysaccharide dissolved in 3% deoxycholate and incubated for 1 h at 568C prior to elution in PBS containing 0.25% deoxycholate. The peaks for the different dextran standards (670 and 150 kDa) are indicated, together with the void volume (V0) and the total volume (Vt). RFU, relative fluorescence units.


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should concentrate on the role of this exopolysaccharide in the pathogenesis of melioidosis. The isolated MAb will help to elucidate the role of antibodies against this structure in the defense of B. pseudomallei in experimental models. In this context, the possible role of the B. pseudomallei exopolysaccharide as a protection-inducing antigen needs to be investigated. ACKNOWLEDGMENTS We thank S. Bhakdi from the Institute of Medical Microbiology at the Medical School in Mainz, Germany; T. Dharakul from the Division of Immunology at the Siriraj Hospital in Bangkok, Thailand; and W. Ba¨r from Cottbus, Germany, all for kindly providing serum specimens from melioidosis patients. We especially thank D. Bitter-Suermann for his continuous interest and support, both of which made this study possible. REFERENCES 1. Baeck, L., N. Hoiby, J. B. Hertz, and F. Espersen. 1985. Interactions between Limulus amoebocyte lysate and soluble antigens from Pseudomonas aeruginosa and Staphylococcus aureus studied by quantitative immunoelectrophoresis. J. Clin. Microbiol. 22:229–237. 2. Chaowagul, W., N. J. White, D. A. B. Dance, Y. Wattanagoon, P. Naigowit, T. M. E. Davis, S. Looareesuwan, and N. Pitakwatchara. 1989. Melioidosis: a major cause of community acquired septicemia in Northeastern Thailand. J. Infect. Dis. 159:890–899. 3. Costerton, J. W., and R. T. Irwin. 1981. The bacterial glycocalyx in nature and disease. Annu. Rev. Microbiol. 35:299–324. 4. Cravitz, L., and W. R. Miller. 1950. Immunologic studies with Malleomyces mallei and Malleomyces pseudomallei. J. Infect. Dis. 86:52–62. 5. Dance, D. A. B. 1989. Identification of Pseudomonas pseudomallei in clinical practice: use of simple screening tests and API 20 NE. J. Clin. Pathol. 42:645–648. 6. Dance, D. A. B. 1990. Melioidosis. Rev. Med. Microbiol. 1:143–150. 7. Dance, D. A. B. 1991. Pseudomonas pseudomallei: danger in the paddy fields. Trans. R. Soc. Trop. Med. Hyg. 85:1–3. 8. Dance, D. A. B. 1991. Melioidosis: the tip of the iceberg? Clin. Microbiol. Rev. 4:52–60. 9. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350–356. 10. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual, p. 522–523.

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