Molecular and physical characterization of Burkholderia mallei O antigens - PubMed (original) (raw)

Molecular and physical characterization of Burkholderia mallei O antigens

Mary N Burtnick et al. J Bacteriol. 2002 Feb.

Abstract

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.

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Figures

FIG. 1.

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.

FIG. 2.

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 _Bam_HI and _Kpn_I cleavage sites used for subcloning are shown. Two additional _Bam_HI 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.

FIG. 3.

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.

FIG. 4.

FIG. 4.

Serum bactericidal assays with B. mallei strains. (A) Thirty percent NHS killing assay in which viable counts were determined at the 2-, 4-, 8-, and 18-h time points. B. pseudomallei 1026b (▴), B. mallei ATCC 23344 (⧫), and E. coli HB101 (•). (B) Thirty percent NHS killing assay in which viable counts were determined following a 2-h incubation at 37°C. Control tubes containing phosphate-buffered saline (PBS) are shown as white bars, and experimental tubes containing 30% NHS are shown as gray bars. Error bars indicate standard deviations.

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References

    1. Brett, P. J., and D. E. Woods. 1996. Structural and immunological characterization of Burkholderia pseudomallei O-polysaccharide-flagellin protein conjugates. Infect. Immun. 64:2824–2828. - PMC - PubMed
    1. Bryan, L., S. Wong, D. Woods, D. Dance, and W. Chaoagul. 1994. Passive protection of diabetic rats with antisera specific for the polysaccharide portion of lipopolysaccharide from Pseudomonas pseudomallei. Can. J. Infect. Dis. 5:170–178. - PMC - PubMed
    1. Centers for Disease Control and Prevention. 2000. Laboratory acquired human glanders—Maryland May 2000. Morb. Mortal. Wkly. Rep. 49:532–533. - PubMed
    1. DeShazer, D., P. J. Brett, R. Carlyon, and D. E. Woods. 1997. Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: isolation of motility mutants and molecular characterization of the flagellin structural gene. J. Bacteriol. 179:2116–2125. - PMC - PubMed
    1. DeShazer, D., P. J. Brett, and D. E. Woods. 1998. The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence. Mol. Microbiol. 30:1081–1100. - PubMed

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