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Journal of Bacteriology, February 2002, p . 849-852, Vol . 184, No . 3
Molecular and Physical Characterization of Burkholderia mallei O Antigens
Mary N . Burtnick, Paul J . Brett, and Donald E . Woods*
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada T2N 4N1
Received 1 August 2001/
Accepted 6 November 2001
Burkholderia mallei lipopolysaccharide (LPS) has been previously shown to cross-react with polyclonal antibodies raised against B . pseudomallei LPS; however, we observed that B . mallei LPS does not react with a monoclonal antibody (Pp-PS-W) specific for B . pseudomallei O polysaccharide (O-PS) . In this study, we identified the O-PS biosynthetic gene cluster from B . mallei ATCC 23344 and subsequently characterized the molecular structure of the O-PS produced by this organism .
Burkholderia mallei is a gram-negative bacterium responsible for a disease known as glanders in solipeds and occasionally in humans (3, 8, 13) . The factors involved in the pathogenesis of B . mallei infection remain relatively poorly defined at the molecular level . A previous study that identified a polysaccharide gene cluster in B . mallei showed that B . mallei lipopolysaccharide (LPS) cross-reacts with polyclonal antibodies raised against the LPS of Burkholderia pseudomallei, a closely related organism responsible for a disease known as melioidosis (6) . In the present study, we investigated the LPS profiles of B . mallei strains, identified the gene cluster responsible for O polysaccharide (O-PS) biosynthesis in B . mallei ATCC 23344, and determined the physical structure of the B . mallei ATCC 23344 O-PS . Additionally, we showed that the O-PS moiety of B . mallei LPS is required for resistance to the bactericidal action of serum . Finally, we identified the presence of insertion sequences in two strains of B . mallei that disrupt the expression of O-PS .
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Analysis of LPS profiles of B . mallei strains.
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The strains and plasmids used in this study are shown in Table 1 . The first goal of this study was to assess the LPS profiles of B . mallei strains . Initially, we performed Western blot analysis of B . mallei ATCC 23344 whole-cell lysates with polyclonal rabbit sera raised against a B . pseudomallei bovine serum albumin (BSA)-O-PS conjugate as well as with a B . pseudomallei O-PS-specific MAb (Pp-PS-W) according to a previously described protocol (1, 2) . As shown in Fig . 1A, B . mallei ATCC 23344 reacted with the anti-LPS polyclonal sera, resulting in a typical LPS banding pattern; however, the B . pseudomallei O-PS-specific MAb (Pp-PS-W) did not react . This indicated that differences exist between B . mallei and B . pseudomallei O-PS . We further assessed the LPS profiles of 10 different B . mallei strains (Fig . 1B) . By using Western blot analysis, we showed that 8 of the 10 strains assessed bound the anti-LPS polyclonal sera and displayed typical LPS banding patterns . In contrast, however, two strains, NCTC 120 and ATCC 15310, did not bind the anti-LPS polyclonal sera, as indicated by the absence of bands (Fig . 1B) . In order to confirm that the O-PS moiety was absent rather than a different type of O-PS, silver stain analysis was employed . Figure 1C shows the silver stain results confirming that both of these strains lacked O-PS moieties .
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TABLE 1 . Bacterial strains and cosmids or plasmids used in this study
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FIG . 1 . (A) Western blot analysis of B . mallei ATCC 23344 . Proteinase K-treated whole-cell lysates were used . In lane 1, the primary antibody used was a 1/2,000 dilution of polyclonal antisera raised against a B . pseudomallei BSA-O-PS conjugate, and in lane 2, the primary antibody used was a 1/2,000 dilution of the B . pseudomallei O-PS-specific MAb (Pp-PS-W) . (B) Western blot profiles of proteinase K-treated whole-cell lysates of B . mallei strains . The primary antibody used was polyclonal sera raised against a B . pseudomallei BSA-O-PS conjugate . Lanes: 1, NCTC 120; 2, NCTC 10248; 3, NCTC 10229; 4, NCTC 10260; 5, NCTC 10247; 6, ATCC 23344; 7, NCTC 3708; 8, NCTC 3709; 9, ATCC 10399; and 10, ATCC 15310 . (C) Silver stain analysis of proteinase K-treated whole-cell lysates of B . mallei strains . Lanes: 1, NCTC 120; 2, NCTC 10248; 3, NCTC 10229; 4, NCTC 10260; 5, NCTC 10247; 6, ATCC 23344; 7, NCTC 3708; 8, NCTC 3709; 9, ATCC 10399; and 10, ATCC 15310.
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Identification and characterization of B . mallei ATCC 23344 O-PS biosynthetic gene cluster.
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In order to investigate the genes responsible for O-PS biosynthesis in B . mallei, we constructed a cosmid library by using B . mallei ATCC 23344 genomic DNA and the cosmid pScosBC1 by using a previously described protocol (12) . Colony hybridizations were then performed with a 1.1-kb DNA fragment containing the recently identified B . mallei wbiA gene (P . Brett, M . Burtnick, and D . Woods, unpublished data) . Six positive cosmid clones were obtained . Based on the BamHI-KpnI restriction patterns obtained, two cosmid clones, 1C3 and 2B5, were predicted to harbor the entire B . mallei O-PS gene cluster . Sequence analysis resulted in 19,918 bp of contiguous sequence containing the entire B . mallei O-PS biosynthetic gene cluster with an IS407-like insertion sequence element at the 3' end .
The first 18,738 bp of the B . mallei DNA sequence contained 16 predicted ORFs that were identical to those previously defined as the O-PS biosynthetic gene cluster in B . pseudomallei (Fig . 2) (5) . Sequence alignment of the B . pseudomallei and B . mallei O-PS biosynthetic regions revealed 99% identity at the nucleotide level . The genes comprising the B . mallei O-PS biosynthetic operon were named as per the identical genes found in B . pseudomallei (5) .
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FIG . 2 . Restriction and genetic maps of the B . mallei O-PS biosynthetic gene cluster . (A) Restriction map . The horizontal line represents the B . mallei DNA insert of cosmid 2B5 . The locations of BamHI and KpnI cleavage sites used for subcloning are shown . Two additional BamHI sites at the 5' and 3' ends of 2B5, which were part of the pScosBC1 vector, are not shown . (B) Genetic map . The location and direction of transcription of the genes are represented by arrows, and the gene names are shown below . The gray arrows indicate the genes involved in O-PS biosynthesis based on homology to the B . pseudomallei O-PS biosynthetic operon.
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Physical characterization of B . mallei O-PS moieties.
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In order to structurally analyze the B . mallei O-PS structure, it was necessary to construct a B . mallei strain unable to produce capsular polysaccharide (CPS), because CPS copurifies with LPS . The suicide vector pGSV3008 was employed as previously described to construct B . mallei PB100, a derivative of ATCC 23344 that does not produce CPS (6) . The O-PS was purified as previously described for B . pseudomallei . Figure 3 shows 13C nuclear magnetic resonance (13C-NMR) analysis (Complex Carbohydrate Research Center, University of Georgia, Athens) results demonstrating that the B . mallei O-PS backbone is similar to that previously described for B . pseudomallei O-PS, a heteropolymer of repeating D-glucose and L-talose (9, 10) . However, changes are apparent in the O-acetylation pattern of the B . mallei L-talose residue in comparison to that of B . pseudomallei . Similar to B . pseudomallei O-PS, B . mallei O-PS demonstrates the presence of O-acetyl or O-methyl substitutions at the 2' position of the talose residue . In contrast, B . mallei O-PS is devoid of an O-acetyl group at the 4' position of the talose residue . Thus, the structure of B . mallei O-PS is best described as 3)-ß-D-glucopyranose-(1,3)-6-deoxy- -L-talopyranose-(1-, in which the talose residue contains 2-O-methyl or 2-O-acetyl substituents . Recent studies indicate that the presence of 4-O-acetyl groups on the talose residues of B . pseudomallei O-PS is due to an O-acetylation locus unlinked to the previously described O-PS biosynthetic operon (Brett et al., unpublished) . If this is the case, then the unlinked locus responsible for O-acetylation is either not present or is nonfunctional in B . mallei strains . The presence or absence of O-acetyl groups on the O-PS moieties may have consequences when O-PS is considered as a component of a vaccine that protects against both B . mallei and B . pseudomallei.
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FIG . 3 . 13C-NMR analysis of B . mallei PB100 O-PS . (A) O-Acetyl peaks are indicated by stars . (B) Expanded view of the region running from 100 to 60 ppm . (C) Structure of B . pseudomallei and B . mallei O-PS . In B . pseudomallei, in 33% of the talose residues, R' = O-methyl and R" = O-acetyl, and in 66% of the talose residues, R' = O-acetyl and R" = OH . In B . mallei, R' = O-acetyl or O-methyl and R" = OH.
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B . mallei survives in 30% NHS, and serum-sensitive strains lack the O-PS moiety of LPS.
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The ability of B . mallei ATCC 23344 to grow in the presence of 30% normal human serum (NHS) was initially assessed with a serum bactericidal assay (5) in which viable counts were determined at 2, 4, 8, and 18 h . B . mallei ATCC 23344 was shown to survive in the presence of 30% NHS over the course of 18 h (Fig . 4A) . Serum-resistant B . pseudomallei 1026b and serum-sensitive Escherichia coli HB101 were employed as controls .
In order to assess the role of B . mallei O-PS in serum resistance, NHS bactericidal assays (5) were performed with B . mallei ATCC 23344 and B . mallei NCTC 120 and ATCC 15310, the two strains lacking O-PS . B . mallei ATCC 23344 remained resistant to the killing action of 30% NHS, while NCTC 120 and ATCC 15310 were completely killed following a 2-h incubation in 30% NHS (Fig . 4B) . The other seven B . mallei strains used in this study possessed intact O-PS moieties and were resistant to the bactericidal action of 30% NHS (data not shown) . These results suggested that B . mallei O-PS moieties play a crucial role in the serum resistance of this organism: this correlates well with previous studies demonstrating that B . pseudomallei O-PS is required for serum resistance (5) .
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Identification of insertion sequence IS407 in the O-PS biosynthetic gene clusters of B . mallei NCTC 120 and ATCC 15310.
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In order to determine if the O-PS biosynthetic gene clusters of NCTC 120 and ATCC 15310 had been disrupted, we chose to individually PCR amplify each gene present in this cluster . Deoxyoligonucleotide primers were designed outside of the 5' and 3' ends of each gene . B . mallei ATCC 23344 chromosomal DNA was used a control as an indicator of the size of a wild-type copy of each gene . Alterations were observed in the wbiE PCR product from NCTC 120 and in the wbiG PCR product from ATCC 15310 . The PCR products obtained in both cases were approximately 1.5 kb larger than those obtained with ATCC 23344 genomic DNA (data not shown) . Cloning and sequence analysis of the NCTC 120 wbiE and ATCC 15310 wbiG PCR products revealed the presence of insertion sequences within these two genes . In NCTC 120, an IS407-like element was located after nucleotide 13615 of the O-PS operon in the wbiE gene . In ATCC 15310, an IS407-like element was located following nucleotide 15107 of the O-PS operon in the wbiG gene . It is likely that the presence of insertion elements in the O-PS biosynthetic gene clusters of B . mallei NCTC 120 and ATCC 15310 is responsible for the loss of expression of O-PS in these two strains . DeShazer et al . have previously demonstrated the presence of an IS407-like element (termed "IS407A") at the 3' end of the CPS gene cluster and have shown that this element is active in B . mallei (6) . The data presented in this paper certainly support the view that IS407 is functionally active in B . mallei .
This work was funded by the Department of Defense contract no . DAMD 17-98-C-8003 and CIHR MOP 31343 . M.N.B . is the recipient of an Alberta Heritage Foundation for Medical Research Studentship Award .
We are grateful to Patricia Baker and Francois Becotte for excellent technical assistance . We thank David DeShazer for providing us with the plasmid pGSV3008 .
* Corresponding author . Mailing address: Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, 3330 Hospital Dr . NW, Calgary, Alberta, Canada T2N 4N1 . Phone: 403-220-2564 . Fax: 403-283-5241 . E-mail: woods{at}ucalgary.ca .
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