INFECTION AND IMMUNITY, Feb. 2011, p. 961–969 0019-9567/11/$12.00 doi:10.1128/IAI.01023-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 2
Burkholderia thailandensis oacA Mutants Facilitate the Expression of Burkholderia mallei-Like O Polysaccharides䌤 Paul J. Brett,1* Mary N. Burtnick,1 Christian Heiss,2 Parastoo Azadi,2 David DeShazer,3 Donald E. Woods,4 and Frank C. Gherardini5 Department of Microbiology and Immunology, University of South Alabama, Mobile, Alabama 366881; Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia 306022; Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 217023; Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta, T2N 4N1, Canada4; and Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 598405 Received 21 September 2010/Returned for modification 23 October 2010/Accepted 19 November 2010
Previous studies have shown that the O polysaccharides (OPS) expressed by Burkholderia mallei are similar to those produced by Burkholderia thailandensis except that they lack the 4-O-acetyl modifications on their 6-deoxy-␣-L-talopyranosyl residues. In the present study, we describe the identification and characterization of an open reading frame, designated oacA, expressed by B. thailandensis that accounts for this phenomenon. Utilizing the B. thailandensis and B. mallei lipopolysaccharide (LPS)-specific monoclonal antibodies Pp-PS-W and 3D11, Western immunoblot analyses demonstrated that the LPS antigens expressed by the oacA mutant, B. thailandensis ZT0715, were antigenically similar to those produced by B. mallei ATCC 23344. In addition, immunoblot analyses demonstrated that when B. mallei ATCC 23344 was complemented in trans with oacA, it synthesized B. thailandensis-like LPS antigens. To elucidate the structure of the OPS moieties expressed by ZT0715, purified samples were analyzed via nuclear magnetic resonance spectroscopy. As predicted, these studies demonstrated that the loss of OacA activity influenced the O acetylation phenotype of the OPS moieties. Unexpectedly, however, the results indicated that the O methylation status of the OPS antigens was also affected by the loss of OacA activity. Nonetheless, it was revealed that the LPS moieties expressed by the oacA mutant reacted strongly with the B. mallei LPS-specific protective monoclonal antibody 9C1-2. Based on these findings, it appears that OacA is required for the 4-O acetylation and 2-O methylation of B. thailandensis OPS antigens and that ZT0715 may provide a safe and cost-effective source of B. mallei-like OPS to facilitate the synthesis of glanders subunit vaccine candidates. available for immunization against the disease. Due to the high risk of aerosol infection and the potential for misuse of this organism as an agent of biological warfare and terrorism, B. mallei is currently listed as a select agent by the Centers for Disease Control and Prevention (CDC) (27, 40). Several studies have demonstrated that B. mallei expresses a number of important virulence determinants that are required for survival in animal models of infection such as mice, hamsters, and miniature horses (21). Included among these are a quorum sensing system, an animal pathogen-like type III secretion system, the VirAG two-component regulatory system, the cluster 1 type VI secretion system, and a capsular polysaccharide (5, 15, 29, 38, 39). In addition, studies in our lab and others indicate that the O-polysaccharide (OPS) component of B. mallei lipopolysaccharide (LPS) is both a virulence determinant and a protective antigen (12, 22, 37). Virulent isolates of B. mallei, whether of human or veterinary origin, all appear to express smooth LPS phenotypes (12, 22). Studies conducted in 1925 by Stanton and Fletcher demonstrated that B. mallei NCTC 120, now recognized as a rough isolate, was avirulent in both equine and rabbit models of infection (22, 36). More recently, we have shown that B. mallei strains, including NCTC 120, expressing rough LPS phenotypes are exquisitely sensitive to the bactericidal effects of normal human serum in comparison to those expressing a smooth phenotype (12). Addition-
Burkholderia mallei is a facultative intracellular, Gram-negative bacillus that causes glanders in humans and animals. This zoonotic pathogen is an obligate animal parasite that is primarily responsible for disease in horses, mules, and donkeys (19, 25, 35, 43). In Asia, the Middle East, Africa, and South America, where glanders remains endemic, chronically infected horses are the only known reservoir of this host-adapted pathogen (22). Disease in equines presents as chronic or acute illnesses characterized by lung involvement, ulcerative nasal/ tracheal lesions, and visceral abscess formation. Although rare, human infections are thought to be acquired via the inoculation of mucocutaneous tissues with aerosols or secretions from diseased animals. The clinical progression of human glanders is similar to that observed in solipeds and may manifest as chronic or acute localized infections, acute pulmonary infections, or fulminating septicemias. Diagnosis and treatment of disease can be challenging, and in the absence of chemotherapeutic intervention, human glanders is invariably fatal (2, 18, 41). At present, there are no human or veterinary vaccines
* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of South Alabama, 5851 USA Drive North, Mobile, AL 36688. Phone: (251) 414-8179. Fax: (251) 460-7931. E-mail:
[email protected]. 䌤 Published ahead of print on 29 November 2010. 961
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BRETT ET AL. TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid
Reference or source
Relevant characteristic(s)
E. coli strains TOP10 S17-1
High-efficiency transformation strain: Gms Zeos Mobilizing strain (expresses RP4 tra genes): Kms Gms Pms Zeos
Invitrogen Simon
B. thailandensis strains E264 DW503 ZT0697 ZT0715 ZT1475 ZT0715C ZT0715T
Type strain (environmental isolate) ATCC 700338 (E264) derivative; ⌬(amrR-oprA): Pmr Gms Zeos DW503::pZT0697: Pmr Zeor Gms DW503::pZT0715: Pmr Zeor Gms DW503::pZT1475: Pmr Zeor Gms ZT0715 (pUCP31T): Gmr Pmr Zeor ZT0715 (pCToacA): Gmr Pmr Zeor
7 10 This This This This This
B. mallei strains ATCC 23344 M23344C M23344T
Type strain (human isolate): Pmr Kms ATCC 23344 (pBHR2): Kmr Pmr ATCC 23344 (pBToacA): Kmr Pmr
43 This study This study
Plasmids pZSV pUCP31T pBHR2 pZT0697 pZT0715 pZT1475 pCToacA pBToacA
Mobilizable suicide vector: Zeor Mobilizable broad host range vector: Gmr Mobilizable broad host range vector: Kmr pZSV containing a 488-bp PCR product internal to BTH_II0697: Zeor pZSV containing a 501-bp PCR product internal to BTH_II0715: Zeor pZSV containing a 561-bp PCR product internal to BTH_I1475: Zeor pUCP31T containing a 1,264-bp PCR product encompassing BTH_II0715: Gmr pBHR2 containing a 1,264-bp PCR product encompassing BTH_II0715: Kmr
11 30 29 This This This This This
ally, studies by Trevino et al. indicate that murine monoclonal antibodies (MAbs) specific for B. mallei OPS are capable of passively immunizing mice against a lethal aerosol challenge (37). Because of these attributes, OPS is considered to be a promising component of vaccine candidates for immunoprophylaxis against glanders. Previous studies have shown that the OPS moieties expressed by B. thailandensis (nonpathogenic saprophyte) and Burkholderia pseudomallei (etiologic agent of melioidosis) are unbranched heteropolymers, consisting of disaccharide repeats having the structure -3)--D-glucopyranose-(1-3)-6-deoxy-␣-Ltalopyranose-(1-, in which ⬃33% of the 6-deoxy-␣-L-talopyranose (L-6dTalp) residues possess 2-O-methyl and 4-O-acetyl substitutions while the remainder of the L-6dTalp residues bear only 2-O-acetyl modifications (6, 7, 12, 24). B. mallei expresses OPS antigens that are structurally similar to those expressed by B. thailandensis and B. pseudomallei strains, except that their L-6dTalp residues lack acetyl modifications at the O-4 position (12). This structural difference likely explains the ability to generate MAbs specific for B. thailandensis/B. pseudomallei or B. mallei OPS antigens as well as the presence of B. mallei-specific bacteriophages recognizing smooth and not rough LPS strains (1, 22, 42). Curiously, B. mallei isolates appear to be capable only of expressing a restricted repertoire of structurally diverse OPS antigens. It has even been suggested that virulent isolates of B. mallei can be defined by one serotype (22). At present, the significance of these observations with regard to virulence and evasion of host immune responses remains to be defined. Using a combination of bioinformatic, molecular genetic, immunological, and physicochemical approaches, we set out to
study study study study study
study study study study study
identify the gene(s) responsible for the 4-O acetylation of B. thailandensis OPS moieties. By doing so, it was anticipated that B. thailandensis OPS mutants lacking these modifications would serve as a safe and cost-effective source of B. mallei-like OPS for the development of novel glanders vaccine candidates. In the present study, we describe the identification and characterization of an open reading frame (ORF), designated oacA, expressed by B. thailandensis that is involved in the modification of OPS antigens synthesized by this organism. In addition, we demonstrate that inactivation of oacA results in the production of OPS moieties that are antigenically similar to those expressed by B. mallei. MATERIALS AND METHODS Bacterial strains, growth conditions, and reagents. The bacterial strains used in this study are shown in Table 1. Escherichia coli and B. thailandensis were grown at 37°C on LB-Lennox (LBL; Difco) agar or in LBL broth; B. mallei was grown at 37°C on LBL agar or in LBL broth supplemented with 4% glycerol (LB4G). When appropriate, kanamycin (Km) was used at 25 g/ml for E. coli and 5 g/ml for B. mallei; gentamicin (Gm) was used at 15 g/ml for E. coli and 25 g/ml for B. thailandensis; Zeocin (Zeo; Invitrogen) was used at 25 g/ml for E. coli and 100 g/ml for B. thailandensis. Bacterial stocks were maintained at ⫺80°C as 20% glycerol suspensions. All studies utilizing viable B. mallei were conducted in a CDC select agent-certified biosafety level 3 containment facility. Unless stated otherwise, all reagents were purchased from Sigma. Bioinformatics. WbiA paralogs/orthologs encoded in the genomes of B. thailandensis E264, B. pseudomallei K96243, and B. mallei ATCC 23344 were identified using the ERGO bioinformatics suite (Integrated Genomics Inc.). Pattern-based searches were conducted using two conserved motifs (VXXF FXXSG and WXLXXEXXXY) that are indicative of a family of integral membrane transacylases that encompasses this previously characterized 2-Oacetyltransferase (6, 32). Recombinant DNA techniques. DNA manipulations were performed using standard methods. Restriction enzymes were purchased from Promega or New
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BURKHOLDERIA THAILANDENSIS OacA
TABLE 2. Oligonucleotide primers used in this study Primer
Sequence (5⬘–3⬘)a
BT0697-F2 .............GTACGCGAATTCCTGACGTTCCCGTTCGCATGG BT0697-R2.............GTACGCGCTAGCTACGACGTGAAGAATTGCAGG BT0697-MF2..........TATGGCCATTCGCTCGATCCGAGCG BT0715-F2 .............GTACGCGAATTCTTCCGTTCTGGATTCGACGTGC BT0715-R2.............GTACGCGCTAGCTGAGGCACATTCCTGCAAGAGC BT0715-MF2..........GCATTTCAGGCTACGTCATCACGG BT0715-F3 .............GTACGCAAGCTTCAGCGATGTGGCAAAATCCGTCC BT0715-R3.............GTACGCGAATTCACATGCTGCTCCGGCCGACATCG BT0715-F4 .............GTACGCGAATTCCAGCGATGTGGCAAAATCCGTCC BT0715-R4.............GTACGCTCTAGAACATGCTGCTCCGGCCGACATCG BT1475-F2 .............GTACGCGAATTCGGACCATGCGTTTCCACTTGG BT1475-R2.............GTACGCGCTAGCAATGCCGGATTGATGGTCTGG BT1475-MF2..........CGGGTTGCTTCGTCTGCTGTTCGCG MB1ori-P1 .............GAAGATCCTTTGATCTTTTCTACGG a
Restriction sites in the linker regions are underlined.
England Biolabs and used according to the manufacturer’s instructions. PCR was performed using the Expand high-fidelity PCR system (Roche Applied Science). PCR and restriction-digested products were purified using a QIAquick gel extraction kit (Qiagen). Ligation reactions were performed using a Fast-Link quick ligase kit (Epicentre Technologies) or T4 DNA ligase (New England Biolabs). Plasmids were purified using a QIAprep spin miniprep kit (Qiagen). Genomic DNA was purified using a Wizard genomic DNA purification kit (Promega). Chemically competent E. coli TOP10 cells were transformed in accordance with the manufacturer’s instructions (Invitrogen). DNA sequencing was performed by ACGT Inc. (Wheeling, IL). Construction of B. thailandensis mutants. Plasmids used in this study are described in Table 1. Oligonucleotide primers used in this study are shown in Table 2. Internal fragments of BTH_II0715, BTH_II0697, and wbiA (BTH_I1475) were PCR amplified from B. thailandensis E264 genomic DNA using the BT0715-F2/R2, BT0697-F2/R2, and BT1475-F2/R2 primer pairs, respectively. The DNA fragments obtained were cloned into the EcoRI and XbaI sites of pZSV, a Zeocin-resistant mobilizable suicide vector. The resulting constructs, pZT0715, pZT0697, and pZT1475, were used to insertionally inactivate BTH_II0715, BTH_II0697, and wbiA in B. thailandensis DW503 essentially as previously described (11). Site-specific recombination of the plasmids into the DW503 chromosome was verified via PCR using gene-specific upstream primers (BT0715-MF2, BT0697-MF2, and BT1475-MF2) in combination with a vectorspecific primer (MB1ori-P1). The mutant strains were designated ZT0715, ZT0697, and ZT1475, respectively. Complementation of B. thailandensis ZT0715 and B. mallei ATCC 23344. For complementation of B. thailandensis, the BTH_II0715 ORF was PCR amplified from B. thailandensis E264 genomic DNA using the BT0715-F3/BT0715-R3 primer pair. The DNA fragment obtained was cloned into the HindIII and EcoRI sites of pUCP31T, resulting in plasmid pCToacA. The complementation and control plasmids were mobilized into B. thailandensis ZT0715 essentially as previously described (6). For complementation of B. mallei, the BTH_II0715 ORF was PCR amplified from B. thailandensis E264 genomic DNA using the BT0715-F4/BT0715-R4 primer pair. The DNA fragment obtained was cloned into the EcoRI and XbaI sites of pBHR2, resulting in plasmid pBToacA. The complementation and control plasmids were mobilized into B. mallei ATCC 23344 as previously described (13, 31). Bacteriophage E125 sensitivity testing. Bacteriophage E125 was propagated and used for sensitivity testing essentially as previously described (42). Briefly, ⬃102 PFU of E125 was added to saturated cultures of B. mallei, and the mixture was incubated at 25°C for 20 min, following which molten LBL top agar containing 4% glycerol was added. The mixture was immediately poured onto an LBL plate containing 4% glycerol and incubated overnight at 37°C. Bacteria were considered to be sensitive to E125 if they formed plaques under these conditions and resistant if they did not. Data were analyzed using GraphPad Prism 5 (Graphpad Software Inc., San Diego, CA). Statistical differences were calculated using an unpaired Student t test with the significance set at P ⬍ 0.05. SDS-PAGE, silver staining, and Western immunoblot analysis. Whole-cell lysates were prepared from a variety of B. thailandensis and B. mallei strains. For silver staining, 0.5-ml samples of the overnight bacterial cultures were pelleted, resuspended in 1⫻ Tris-glycine-SDS sample buffer (200 l; Invitrogen), boiled for 10 min, and then treated with proteinase K (10 l of a 5-mg/ml stock solution) for 1 h at 60°C. Following this, the treated lysates were separated on 12% express gels (ISC BioExpress) and the LPSs were visualized as previously described (5, 17). For immunoblot analyses, the treated lysates were separated on 12% express
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gels (ISC BioExpress) and then electrophoretically transferred to nitrocellulose membranes. Immunoblotting was then performed at room temperature as follows: membranes were blocked with 3% skim milk in Tris-buffered saline (TBS) (20 mM Tris, 500 mM NaCl, pH 7.5) for 60 min, followed by incubation for 1 h with 1/200 to 1/1,000 dilutions of the primary antibodies diluted in TBS. The membranes were washed three times with TBS, followed by incubation for 1 h with a 1/2,000 dilution of the secondary antibodies diluted in TBS. The membranes were washed three more times with TBS, and the blots were visualized using the HRP color development solution (Bio-Rad) in accordance with the manufacturer’s instructions. The primary antibodies used were the rabbit polyclonal antiserum (raised against a B. pseudomallei OPS-bovine serum albumin [BSA] glycoconjugate) (12), the B. thailandensis/B. pseudomallei LPS-specific MAb Pp-PS-W (9), the B. mallei LPS-specific MAbs 3D11 and 9C1-2 (Research Diagnostics, Inc.) (42), and the mouse polyclonal antiserum (raised against a B. thailandensis ZT0715 OPS-based glycoconjugate). The secondary antibodies used were anti-rabbit IgG or anti-mouse IgG horseradish peroxidase conjugates. LPS and OPS purification. LBL broth inoculated with B. thailandensis ZT1475 or ZT0715 was incubated overnight at 37°C with vigorous shaking. Cell pellets were subsequently obtained by centrifugation and extracted using a modified hot aqueous-phenol procedure (5, 24). Following extraction, the resulting phenol and aqueous phases were combined and dialyzed in distilled water to remove the phenol. The dialysates were then clarified by centrifugation and concentrated by lyophilization. The crude preparations were solubilized (20 mg/ml) in RD buffer (10 mM Tris-HCl [pH 7.5], 1 mM MgCl2, 1 mM CaCl2, 50 g/ml RNase A, and 50 g/ml DNase I) and incubated for 3 h with shaking at 37°C. Proteinase K was then added to a final concentration of 50 g/ml, and the digests were incubated for an additional 3 h at 60°C. The enzymatic digests were clarified by centrifugation, and the supernatants were filter sterilized. LPS was then isolated from the supernatants as precipitated gels following three rounds of ultracentrifugation at 100,000 ⫻ g and 4°C. After the final spin, the gel-like pellets were resuspended in pyrogen-free water and lyophilized. To remove contaminating phospholipids, the lyophilized LPS samples were repeatedly extracted with 90% ethyl alcohol (EtOH). To obtain purified OPS for the nuclear magnetic resonance (NMR) analyses, the LPS samples were solubilized (5 mg/ml) in 2% acetic acid and incubated for 2 h at 100°C (24). The hydrolyzed samples were cooled to room temperature and clarified via centrifugation (10 min at 8,000 ⫻ g), following which the supernatants were carefully removed and lyophilized to concentrate. The crude OPS samples were then solubilized (25 mg/ml) in phosphate-buffered saline (PBS), clarified via centrifugation (10 min at 8,000 ⫻ g), and filter sterilized. The samples were loaded onto Sephadex G-50 columns (40 cm by 2.6 cm) equilibrated with PBS and eluted with the same buffer. Fractions eluting near the void volumes were assayed for carbohydrate using the phenol-sulfuric acid method (16). Appropriate fractions were pooled, extensively dialyzed against distilled water, and lyophilized. NMR spectroscopy. The purified OPS samples were analyzed at the Complex Carbohydrate Research Center (University of Georgia, Athens, GA). NMR experiments were carried out in D2O (99.996% D; Cambridge Isotope Laboratories) on a Varian Inova 600 MHz spectrometer using a 5-mm triple resonance probe, which was kept at a temperature of 25°C. Heteronuclear 1H-13C chemical shift correlations were measured by gradient-enhanced heteronuclear single quantum correlation (gHSQC) spectra in the 1H-detected mode. Standard pulse sequences from Varian were used for all experiments. The spectral width was 3,000 Hz in the proton dimension in all experiments and 24,000 Hz in the carbon dimension in the gHSQC experiments. Chemical shifts are reported in parts per million downfield from internal DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) but were actually measured relative to internal acetone (␦C ⫽ 31.07 ppm and ␦H ⫽ 2.225 ppm). Glycoconjugate synthesis and antibody production. Purified ZT0715 OPS was solubilized at 2.5 mg/ml in PBS and added to a small amber vial. To each milliliter of the OPS solution was added 6 mg (⬃30 mM) of sodium metaperiodate. Once the crystals had dissolved by gentle agitation, the reaction mixture was incubated for 30 min at room temperature in the dark. To remove any excess oxidizing agent, the reaction mixture was applied to Zeba Desalt spin columns (Pierce) equilibrated with PBS and the eluate was collected. To facilitate conjugation of the OPS to the carrier protein (SuperCarrier immune modulator; Pierce), the activated OPS was added to a small amber vial. To each milliliter of the OPS solution was added 125 l of the carrier protein in PBS (10 mg/ml stock). Following mixing by gentle agitation, 10 l of a 1 M sodium cyanoborohydride stock was added to each milliliter of the conjugation mixture and the reaction mixture was incubated overnight at room temperature in the dark. To remove any excess reducing agent, the conjugate reaction was applied to a Zeba Desalt spin column equilibrated with PBS and the eluate was collected.
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TABLE 3. Identification of B. thailandensis, B. pseudomallei, and B. mallei wbiA homologsa ERGO annotation O-antigen acetylase Integral membrane acetyltransferase Integral membrane acetyltransferase Acyltransferase family protein O-antigen acetylase Acyltransferase Hypothetical protein O-antigen acetylase
B. thailandensis TIGR ID
B. pseudomallei SANGER ID
B. mallei TIGR ID
BTH_I1858 BTH_I1965
None BPSL2220
None BMA_A1626
BTH_II0626
BPSS1754
BMA_A0415
BTH_II0691
BPSS1687
BMA_A1708
BTH_II0697 BTH_II0700 BTH_II0715 BTH_II2132
BPSS1683 BPSS1681 BPSL1936 BPSS0268
None BMA_A1701 None BMA_A1498
a ERGO annotation, annotation assigned in the ERGO bioinformatics suite database; TIGR ID, locus tag assigned by The Institute for Genomic Research; SANGER ID, locus tag assigned by the Wellcome Trust Sanger Institute.
The glycoconjugate was then stored at ⫺20°C until used for the production of mouse polyclonal antiserum at Cocalico Biologicals using a standard immunization protocol.
RESULTS Identification of putative acetyltransferase genes in B. thailandensis, B. pseudomallei, and B. mallei. We have previously demonstrated that wbiA is required for the 2-O acetylation of B. thailandensis and B. pseudomallei OPS antigens (6). This gene is located within a highly conserved OPS biosynthetic gene cluster expressed by B. thailandensis, B. pseudomallei, and B. mallei (12, 14). Because of this and the absence of 4-O-acetyl modifications associated with B. mallei OPS, we predicted that the gene encoding the 4-O-acetylase activity in B. thailandensis and B. pseudomallei would be physically unlinked to the OPS biosynthetic gene cluster and would either be absent from or nonfunctional in B. mallei. To identify ORFs encoding candidate proteins, the B. thailandensis E264 genome was scanned for paralogs of WbiA. Motif-based searches were performed using two conserved amino acid motifs present in WbiA that are commonly associated with an acyltransferase protein family (PF01757) often involved in the acetylation of bacterial polysaccharides (6, 32). Initial searches using motif 1 (VXXF FXXSG) resulted in the identification of 11 ORFs. Additional searches for the presence of motif 2 (WXLXXEXXXY) reduced this subset to eight candidate genes (Table 3). Once identified, the B. pseudomallei K96243 and B. mallei ATCC 23344 genomes were scanned for orthologs of each of these putative acetyltransferase genes. Seven ORFs were identified in B. pseudomallei, and five ORFs were identified in B. mallei (Table 3). The most promising candidate genes were considered to be those that were present in both B. thailandensis and B. pseudomallei but absent from B. mallei. Based on these criteria, BTH_II0697 and BTH_II0715 were selected for further analysis. Characterization of B. thailandensis BTH_II0697 and BTH_II0715 mutants. To assess the importance of BTH_II0697 and BTH_II0715 expression with respect to OPS modification, the ORFs were mutated in B. thailandensis DW503 by insertional inactivation with pZT0697 and pZT0715, respectively (Table 1). The resulting mutant strains were designated ZT0697 (BTH_II0697 mutant) and ZT0715
(BTH_II0715 mutant). For control purposes, B. thailandensis DW503 (wild type) and ZT1475 (wbiA; 2-O-acetyl mutant) were also used throughout this study (6, 10). To determine the effect(s) of the BTH_II0697 and BTH_II0715 mutations on LPS expression, proteinase K-treated whole-cell lysates were subjected to silver staining. The results demonstrated that all of the mutant and control strains exhibited banding patterns consistent with the expression of smooth LPS species (data not shown). In addition, all four of the B. thailandensis strains were found to react with the anti-B. pseudomallei OPS polyclonal rabbit serum, resulting in characteristic LPS banding patterns. Interestingly, however, the polyclonal antiserum did not react as strongly with the ZT0715 LPS antigens as with the other LPS species (Fig. 1A). Nonetheless, these findings indicated that disruptions in BTH_II0697 and BTH_II0715 did not inhibit LPS expression. To examine the reactivity of the LPS antigens expressed by B. thailandensis ZT0697 and ZT0715 with species-specific MAbs, Western immunoblot analyses were conducted using the previously described B. thailandensis LPS-specific MAb, Pp-PS-W, and the B. mallei LPS-specific MAb, 3D11 (9, 42). As shown in Fig. 1B and C, the DW503 and ZT0697 LPS antigens reacted strongly with Pp-PS-W but not 3D11, whereas in contrast, the ZT0715 LPS reacted strongly with 3D11 but not PS-Pp-W. Consistent with previous observations, ZT1475 LPS did not react with either of the two MAbs (Fig. 1B and C). Since we have previously demonstrated that Pp-PS-W specifically recognizes OPS antigens that are coordinately acetylated at both the O-2 and O-4 positions (6), these findings suggested that disruption of BTH_II0715 resulted in the expression of OPS moieties that were antigenically similar to those of B. mallei OPS and were likely to be devoid of acetyl modifications at the O-4 position of the L-6dTalp residues. In contrast, mutation of BTH_II0697 did not appear to affect LPS expression or MAb reactivity patterns in comparison to the DW503 control. Based on this, we chose to focus further efforts on the characterization of BTH_II0715. Complementation analysis of B. thailandensis ZT0715. To confirm that the mutation in B. thailandensis ZT0715 was nonpolar, we complemented this strain in trans by providing a wild-type copy of BTH_II0715 under the control of a consti-
FIG. 1. Western immunoblot analysis of LPS antigens expressed by wild-type and mutant strains of B. thailandensis. Proteinase K-treated whole-cell lysates were probed with the anti-B. pseudomallei OPS polyclonal rabbit serum (A), the B. thailandensis LPS-specific MAb PpPS-W (B), and the B. mallei LPS-specific MAb 3D11 (C). Strains: DW503 (wild type), ZT0697 (BTH_II0697 mutant), ZT0715 (BTH_II0715 mutant), and ZT1475 (wbiA mutant).
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FIG. 2. Complementation analysis of B. thailandensis ZT0715. Proteinase K-treated whole-cell lysates were probed with the B. thailandensis LPS-specific MAb Pp-PS-W (A) and the B. mallei LPS-specific MAb 3D11 (B). Strains: DW503 (wild-type control), ZT0715C (BTH_II0715 mutant harboring pUCP31T) and ZT0715T (BTH_II0715 mutant harboring pCToacA).
tutive promoter on a broad-host-range plasmid. The resulting strain was designated ZT0715T (BTH_II0715 mutant harboring pCToacA). Western immunoblot analysis was used to compare the reactivity profiles of ZT0715T, ZT0715C (BTH_II0715 mutant harboring pUCP31T), and DW503 LPSs with the B. thailandensis and B. mallei LPS-specific MAbs. As shown in Fig. 2, the DW503 and ZT0715T antigens reacted strongly with Pp-PS-W and not 3D11, whereas the ZT0715C LPS reacted strongly with 3D11 and not Pp-PS-W. Taken together, these findings indicated that the mutation in BTH_II0715 was complementable (nonpolar) and further suggested that we had successfully identified an ORF in B. thailandensis that encoded a protein involved in the 4-O acetylation of the OPS antigens expressed by this species. Based upon our collective findings to this point, we designated this ORF oacA, for O-antigen acetylase A. Complementation analysis of B. mallei ATCC 23344. To determine if the product encoded by the B. thailandensis oacA allele could modify LPS antigens produced by a B. mallei isolate, the gene was expressed in wild-type B. mallei ATCC 23344. To accomplish this, the oacA complementation plasmid, pBToacA, was mobilized into B. mallei ATCC 23344, resulting in strain M23344T. Western immunoblot analysis was utilized to compare the reactivities of the M23344T and M23344C (ATCC 23344 harboring pBHR2) LPS species with the B. thailandensis and B. mallei LPS-specific MAbs. As expected, the control strain M23344C LPS reacted with 3D11 and not Pp-PS-W (Fig. 3A). In contrast, the M23344T LPS reacted with Pp-PS-W and not 3D11 (Fig. 3B). Based on these observations, expression of oacA in B. mallei appeared to convert the OPS antigens to B. thailandensis-like OPS. These results further suggested that oacA encoded a product involved in the 4-O acetylation of B. thailandensis and B. mallei OPS antigens. Bacteriophage protection assays. Previous studies have shown that while B. mallei isolates expressing smooth LPS phenotypes are sensitive to infection with bacteriophage E125, B. pseudomallei and B. thailandensis isolates are not. To account for this phenomenon, it has been suggested that B. mallei might be sensitive to E125 simply because it produces OPS antigens that are devoid of 4-O acetylation modifications (42). To test this hypothesis, we infected B. mallei ATCC
BURKHOLDERIA THAILANDENSIS OacA
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FIG. 3. Complementation analysis of B. mallei ATCC 23344. Proteinase K-treated whole-cell lysates were probed with the B. mallei LPS-specific MAb 3D11 (A) and the B. thailandensis LPS-specific MAb Pp-PS-W (B). Strains: M23344C (ATCC 23344 harboring pBHR2) and M23344T (ATCC 23344 harboring pBToacA).
23344, M23344C, and M23344T with E125 and scored for plaque formation at 24 h postinfection. Interestingly, following the incubation of the bacterium/bacteriophage mixtures, all three strains were shown to be sensitive to E125. More specifically, there was no statistically significant difference in the number of phage plaques observed between the different B. mallei strains (data not shown). Based upon these findings, it appeared that the ability of B. mallei to express B. thailandensis-like OPS antigens did not protect it from infection with the bacteriophage. Spectroscopic analysis of B. thailandensis OPS antigens. To elucidate the structure of OPS moieties expressed by B. thailandensis ZT0715, OPS samples isolated from purified LPS antigens were analyzed using one-dimensional (1-D) 13C, 1-D 1 H, and 2-D 1H-13C gHSQC NMR experiments. For control purposes, a 1-D 13C NMR spectrum was also obtained for purified ZT1475 OPS. Consistent with previous studies, the 13 C NMR spectrum of the ZT1475 OPS sample revealed anomeric carbon signals between 98.0 and 102.3 ppm, one O-acetyl signal at 174.2 ppm (CH3CO), one O-acetyl signal at 20.7 (CH3CO), one O-methyl at 58.8 ppm, and two 6-deoxyhexose methyl signals at 15.4 and 15.7 ppm, indicating that the OPS moieties were devoid of any 2-O-acetyl substitutions (Fig. 4A and 5A) (6). In contrast, and quite unexpectedly, the 13C NMR spectrum of the ZT0715 OPS sample revealed anomeric carbon signals between 98.7 and 102.0 ppm, one O-acetyl signal at 173.7 ppm (CH3CO), one O-acetyl signal at 20.8 (CH3CO), and one 6-deoxyhexose methyl signal at 15.5 ppm, indicating that OPS antigens were devoid of both 4-O-acetyl and 2-Omethyl modifications (Fig. 4B and 5B). Importantly, 1H and gHSQC NMR analyses of the ZT0715 OPS sample also supported these observations (data not shown). Collectively, these results demonstrated that unlike WbiA, OacA influenced both the O acetylation and O methylation status of the OPS antigens expressed by B. thailandensis. Reactivity of ZT0715 with the protective MAb 9C1-2. Previous studies by Trevino et al. have indicated that a number of B. mallei-specific MAbs are capable of passively immunizing mice against a lethal aerosol challenge of B. mallei (37). Since one of the goals of the present study was to identify B. thailandensis
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FIG. 4. 13C NMR spectra of the mutant OPS antigens expressed by B. thailandensis strains ZT1475 (wbiA mutant) (A) and ZT0715 (oacA mutant) (B). OAc. O-acetyl; OMe, O-methyl; Me, methyl.
OPS mutants for use in glanders vaccine development, we were interested in determining if the LPS moieties expressed by ZT0715 could be recognized by the B. mallei LPS-specific protective MAb 9C1-2. Similarly to previous experiments using the 3D11 MAb, immunoblot analyses demonstrated that while the ZT0715C LPS antigens reacted strongly with the 9C1-2 MAb, the LPSs expressed by DW503 and ZT0715T did not (Fig. 6). Building upon these findings, studies were initiated to investigate whether or not polyclonal antiserum raised against ZT0715 OPS would cross-react with LPS antigens expressed by B. mallei. To facilitate these studies, we chemically activated purified ZT0715 OPS, conjugated it to a carrier protein, and immunized mice with the resulting glycoconjugate. When the immune serum raised against the glycoconjugate was used, the results demonstrated that the OPS component was capable of stimulating antibody responses that reacted strongly with LPS antigens expressed by both B. thailandensis ZT0715 and B. mallei ATCC 23344 (Fig. 7). Taken together, these findings indicated that although ZT0715 OPS is not structurally identical to B. mallei OPS, it may still prove useful in the development of novel glycoconjugate vaccine candidates.
DISCUSSION 13
FIG. 5. C NMR spectra of the mutant OPS antigens expressed by B. thailandensis strains ZT1475 (wbiA mutant) (A) and ZT0715 (oacA mutant) (B) expanded between the region of 14 to 22 ppm. OAc, O-acetyl; Me, methyl.
LPS is known to play a key role in the interactions of bacteria with the host immune system. In general, OPS moieties are considered to be the most immunodominant components
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FIG. 6. Reactivity of B. thailandensis ZT0715 LPS antigens with a B. mallei OPS-specific protective MAb. Proteinase K-treated wholecell lysates were probed with the MAb 9C1-2. Strains: DW503 (wildtype control), ZT0715C (oacA mutant harboring pUCP31T), and ZT0715T (oacA mutant harboring pCToacA).
of LPS molecules (26). Moreover, OPS has been identified as both a virulence factor and a protective antigen in many Gramnegative pathogens, including B. pseudomallei and B. mallei (4, 8, 9, 14, 26, 28). The structure of B. thailandensis OPS is identical to that of B. pseudomallei, and it differs from that of B. mallei OPS by the presence of acetyl groups at the O-4 position of the L-6dTalp residues (6, 12, 24). In the present study, we used bioinformatic analyses to identify a locus, designated oacA, that is required for modification of OPS antigens in B. thailandensis. As anticipated, an oacA homolog (BPSL1936) was present in the B. pseudomallei K96243 genome but was absent from the B. mallei ATCC 23344 genome. These findings are consistent with the results of whole-genome comparisons conducted by Kim et al. (20). As indicated by the locus tags, the B. thailandensis and B. pseudomallei oacA alleles (BTH_II0715 and BPSL1936) are located on different chromosomes in each organism. In addition, these genes map to genome locations that are physically unlinked to the previously described gene clusters responsible for OPS biosynthesis. A recent study by Song et al. also indicates that BPSL1936 homologs are absent from all of the B. mallei strains sequenced to date due to genomic deletions and that this ORF is disrupted in B. pseudomallei strain 1655, a clinical isolate from Australia, due to an apparent 2-base-pair insertion (23, 34). It will be interesting to examine the structure and antibody reactivity patterns of the OPS moieties expressed by B. pseudomallei 1655 to assess the phenotypic effects of this mutation if indeed it turns out to be real and not just a sequencing artifact. Previous studies have shown that acetylation patterns are often important for immune responses to bacterial surface carbohydrates and that the loss of acetyl groups from these antigens can have a dramatic effect on antibody recognition (3, 26, 32, 33). For example, Berry et al. have demonstrated that in Neisseria meningitidis, O-acetyl modifications define immunogenic epitopes that are associated with the serogroup A capsular polysaccharide (3). Additionally, it has been reported that mutations disrupting the activity of the Salmonella typhimurium O-antigen acetylase OafA resulted in strains expressing LPS molecules that fail to react with certain LPS-specific
FIG. 7. Reactivity of B. thailandensis ZT0715 (oacA mutant) and B. mallei ATCC 23344 (type strain) proteinase K-treated whole-cell lysates with mouse polyclonal antiserum raised against a ZT0715 OPSbased glycoconjugate.
MAbs (32, 33). Likewise, we have previously shown that the inactivation of WbiA in B. thailandensis and B. pseudomallei results in the expression of OPS moieties that no longer react with the LPS-specific protective MAb Pp-PS-W (6). During the present study, we demonstrated that while the inactivation of oacA in B. thailandensis did not inhibit LPS expression, it did result in the expression of LPS antigens that displayed decreased reactivity with OPS-specific polyclonal antiserum in comparison to both the wild-type and the wbiA mutant strains. This finding suggested that the loss of OacA activity significantly altered the structure of the OPS moieties expressed by ZT0715. Additional results showed that, similar to those of the wbiA mutant, the LPS antigens expressed by the oacA mutant no longer reacted with the Pp-PS-W MAb. This observation suggested that the activities encoded by wbiA and oacA are both required for recognition of OPS by the Pp-PS-W MAb and is consistent with previous studies indicating that Pp-PS-W reacts only with OPS moieties that are coordinately acetylated at both the O-2 and O-4 positions (6, 12). Interestingly, the LPS antigens expressed by the oacA mutant gained reactivity with the B. mallei LPS-specific 3D11 MAb, a finding that was indicative of OPS antigens lacking 4-O-acetyl modifications. Complementation of the oacA mutant confirmed that the antibody reactivity patterns correlated with OacA expression. These findings not only demonstrated that ZT0715 expressed B. mallei-like LPS; they also strongly suggested that oacA encoded an OPS-modifying enzyme required for the 4-O acetylation of B. thailandensis OPS antigens. By expressing oacA in trans, we demonstrated that B. mallei LPS could be converted to B. thailandensis-like LPS. Given that native B. mallei OPS antigens lack 4-O-acetyl groups and that the Pp-PS-W MAb is specific for OPS moieties harboring both O-2- and O-4-acetyl modifications (6, 12), these complementation studies supported that OacA was a 4-O-acetyltransferase. Complementation of B. mallei also allowed us to deter-
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FIG. 8. Characterized activities of the OPS modifying enzymes, OacA and WbiA, expressed by B. thailandensis.
mine whether or not OacA activity influenced the sensitivity of the organism toward bacteriophage E125 infection. The results obtained showed that even though B. mallei 23344T expressed B. thailandensis-like LPS moieties, this was not sufficient to block infectivity by E125. These findings indicated that the binding of this bacteriophage to B. mallei may be more complex than previously anticipated. However, we cannot rule out the possibility that all of the E125 binding sites on the OPS antigens were not blocked by the expression of oacA in B. mallei. Further studies will be required to address this issue. Although the OPS antigens expressed by the B. thailandensis oacA mutant were antigenically similar to those of B. mallei OPS, structural analyses revealed that these moieties lacked both 4-O-acetyl and 2-O-methyl modifications (Fig. 8). While the loss of the 4-O-acetyl groups was expected, the absence of methyl groups at the O-2 position of the L-6dTalp residues was surprising for a number of reasons. First, as previously stated, the only apparent structural difference between B. thailandensis and B. mallei OPS is the lack of 4-O-acetyl modifications on B. mallei OPS moieties. Second, while OacA demonstrates homology to a family of well-characterized acyltransferases, it exhibited no obvious homology to any known methyltransferases. At present, the reason for the absence of the 2-Omethyl groups from the ZT0715 OPS antigens is unclear. It is interesting to speculate that OacA may be a unique bifunctional enzyme that possesses both acetyltransferase and methyltransferase activities. Alternatively, it is possible that 4-Oacetyl groups must be present on the L-6dTalp residues so that the methyltransferase expressed by B. thailandensis can modify the O-2 position. If the latter is the case, it is conceivable that B. thailandensis and B. mallei utilize different mechanisms to O methylate their L-6dTalp residues. Further studies are necessary to more thoroughly investigate this phenomenon. B. mallei OPS has previously been identified as a protective antigen and is considered to be a promising component of subunit-based vaccine candidates (4, 37). The identification and inactivation of oacA in B. thailandensis described herein
have allowed, for the first time, the expression of B. mallei-like OPS antigens in a nonpathogenic background. The OPS moieties expressed by the oacA mutant not only reacted with a B. mallei LPS-specific MAb, 3D11; they were also recognized by the protective MAb, 9C1-2. These observations suggested that the 4-O-acetyl modifications associated with native B. thailandensis OPS antigens likely act to mask an epitope(s) recognized by the 3D11 and 9C1-2 MAbs and that the OPS moieties expressed by the oacA mutant harbor a B. mallei-specific protective epitope. In addition, studies confirmed that polyclonal antiserum raised against ZT0715 OPS reacted strongly with LPS antigens expressed by wild-type B. mallei. Collectively, these findings support that the oacA mutant will be a useful source of B. mallei-like OPS for use in the glycoconjugate vaccine candidates that we are currently developing in our lab. Future studies are planned to determine how the presence or absence of the 2-O-methyl substitutions influences the immunogenicity of glycoconjugates synthesized with OPS antigens derived from native (B. mallei ATCC 23344) or mutant (B. thailandensis ZT0715) sources. In summary, these studies confirm that OacA is an O-antigen modifying enzyme responsible for the phenotypic differences between B. thailandensis and B. mallei OPS moieties. We have presented evidence demonstrating that oacA encodes an OPS modification enzyme that influences both the 4-O-acetyl and 2-O-methyl phenotypes of B. thailandensis OPS antigens. In addition, we have provided evidence that ZT0715 may provide a novel means by which to construct glanders vaccine candidates. Ultimately, however, active immunization/challenge studies will be required to fully test our hypothesis. ACKNOWLEDGMENTS This research was supported in part by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases, the Department of Energy-funded (DE-FG09-93ER-20097) Center for Plant and Microbial Complex Carbohydrates, and lab start-up funds from the University of South Alabama.
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