Molecular Pathogenesis

Development of Signature-Tagged Mutagenesis in Burkholderia pseudomallei To Identify Genes Important in Survival and Pathogenesis

J. Cuccui, A. Easton, K. K. Chu, G. J. Bancroft, P. C. F. Oyston, R. W. Titball, B. W. Wren
DOI: 10.1128/IAI.01240-06

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Organ load in BALB/c mice infected i.n. with 1 × 104 CFU of B. pseudomallei K96243. •, lungs; ○, spleens. Symbols indicate counts per animal, and bars indicate means per group.

  • FIG. 2.
    FIG. 2.

    (A) Attenuating transposon insertion sites in B. pseudomallei K96243. (B) Transposon insertion sites within the capsular polysaccharide biosynthesis region of B. pseudomallei K96243. Transposon insertion sites are indicated by vertical arrows. Genes and regions are not drawn to scale.

  • FIG. 3.
    FIG. 3.

    Binding of monoclonal antibody 4V1H12 to the capsular polysaccharide of B. pseudomallei K96243 and selected transposon derivatives with insertions in the capsular polysaccharide coding region. (A) B. pseudomallei K96243 fluorescence view; (B) fluorescence view superimposed on phase-contrast image; (C and D) mutant 13H8 (wcbB); (E and F) mutant 1B9 (wcbC); (G and H) mutant 1A6 (wcbN).

  • FIG. 4.
    FIG. 4.

    (A) Survival assay of mice infected with 1 × 103 CFU of B. pseudomallei K96243 (▪) or the wcbN mutant (▿). (B) Survival assay of mice infected with 1 × 103 of CFU B. pseudomallei K96243 (▪) or the wcbC mutant (▵).

  • FIG. 5.
    FIG. 5.

    (A) Organ loads obtained from lungs of BALB/c mice infected i.n. with 1 × 103 CFU of wild-type (WT) B. pseudomallei K96243 (▴) or wcbC mutant strain 1B9 (B9) (▵). (B) Organ loads obtained from spleens of BALB/c mice infected i.n. with 1 × 103 CFU of B. pseudomallei K96243 (•) or wcbC mutant strain 1B9 (▵). Each symbol indicates counts from an individual mouse. Solid lines indicate mean values per group, and the broken lines show the limit of detection for this assay. NS, not statistically significant; S, significant (P < 0.05).

  • FIG. 6.
    FIG. 6.

    Growth of B. pseudomallei K96243 and mutant strain 13B11 in M9 minimal medium with and without aromatic compound supplementation. ⧫, B. pseudomallei K96243 in M9 minimal medium; ▴, 13B11 in M9 minimal medium; □, B. pseudomallei K96243 in M9 minimal medium with aromatic compounds; ○, 13B11 in M9 minimal medium with aromatic compounds. The results indicate the means of three experiments each carried out in triplicate. Bars indicate standard deviations. O.D. 600, optical density at 600 nm.

  • FIG. 7.
    FIG. 7.

    Protection assay of BALB/c mice vaccinated with B. pseudomallei mutant strain 13B11. ▪, naive mice; ⋄, mice vaccinated with 1 × 105 CFU of mutant strain 13B11; ▾, mice vaccinated with 1 × 106 CFU of mutant strain 13B11. Mice were challenged with 1 × 103 CFU of B. pseudomallei K96243 at day 35 postvaccination.

  • FIG. 8.
    FIG. 8.

    (A) Organ loads obtained from lungs of BALB/c mice infected i.n. with 6 × 105 CFU of wild-type (WT) B. pseudomallei K96243 (•) or 7.5 × 105 CFU of aroB mutant strain 13B11 (▵). (B) Organ loads obtained from spleens of BALB/c mice infected i.n. with 6 × 105 CFU of wild-type B. pseudomallei K96243 (•) or 7.5 × 105 CFU of aroB mutant strain 13B11 (▵). Solid lines indicate mean values per group, and broken lines show the limit of detection for this assay. S, significant (P < 0.05).

Tables

  • TABLE 1.

    Transposon insertion sites identified in this studya

    MutantDisrupted geneProductPutative functionInsertion distance from start codon (bp)b
    7A6wcbAPutative capsular polysaccharide export proteinCapsular polysaccharide biosynthesis1,301
    13E8wcbAPutative capsular polysaccharide export proteinCapsular polysaccharide biosynthesis713
    13H8wcbBPutative capsular polysaccharide glycosyltransferase biosynthesis proteinCapsular polysaccharide biosynthesis46
    1B9wcbCPutative capsular polysaccharide export proteinCapsular polysaccharide biosynthesis1,035
    8F1wcbCPutative capsular polysaccharide export proteinCapsular polysaccharide biosynthesis1,018
    6E3wcbCPutative capsular polysaccharide export proteinCapsular polysaccharide biosynthesis518
    4A4wcbDPutative ABC transporter transmembrane proteinCapsular polysaccharide biosynthesis961
    JCX2C5wzmPutative capsule polysaccharide export ABC transporter transmembrane proteinCapsular polysaccharide biosynthesis466
    8C3wcbEPutative glycosyltransferaseCapsular polysaccharide biosynthesis196
    13G10wcbEPutative glycosyltransferaseCapsular polysaccharide biosynthesis945
    4H9wcbEPutative glycosyltransferaseCapsular polysaccharide biosynthesis1,464
    5E12wcbEPutative glycosyltransferaseCapsular polysaccharide biosynthesis1,346
    6G8wcbHPutative glycosyltransferaseCapsular polysaccharide biosynthesis625
    8H1wcbHPutative glycosyltransferaseCapsular polysaccharide biosynthesis731
    9G8wcbHPutative glycosyltransferaseCapsular polysaccharide biosynthesis474
    JCX2B10wcbHPutative glycosyltransferaseCapsular polysaccharide biosynthesis75
    4E6wcbIPutative capsular polysaccharide biosynthesis proteinCapsular polysaccharide biosynthesis459
    JCX2B2wcbJPutative capsular polysaccharide biosynthesis proteinCapsular polysaccharide biosynthesis516
    1A6wcbNPutative d-glycero-d-manno-heptose 1,7-bisphosphate phosphataseCapsular polysaccharide biosynthesis258
    13G3wcbQPutative capsular polysaccharide transmembrane export proteinCapsular polysaccharide biosynthesis711
    12F5wcbRPutative type I polyketide synthaseCapsular polysaccharide biosynthesis761
    JCX2A9wcbRPutative type I polyketide synthaseCapsular polysaccharide biosynthesis451
    8A8BPSL0776RecAHomologous recombination, SOS response207
    7G7BPSL2825Putative PabBBiosynthesis of para-aminobenzoate1,352
    9D6serCPhosphoserine aminotransferaseSerine biosynthesis360
    6H5trpGAnthranilate synthase component IITryptophan biosynthesis447
    12C6leuBIsopropylmalate dehydrogenaseLeucine biosynthesis769
    8H10leuBIsopropylmalate dehydrogenaseLeucine biosynthesis899
    13B11aroBDehydroquinate synthaseChorismate biosynthesis532
    7A1BPSL3147Putative lipoprotein39.16% amino acid identity to Shigella flexneri VacJ lipoprotein618
    6H4BPSL1039Putative ABC transporterPutative transmembrane ABC transporter1,293
    7G1BPSL0634Putative oxidoreductaseEnergy production and conversion2,306
    6A8BPSL0634Putative oxidoreductaseEnergy production and conversion3,191
    13D4ppsAPutative phosphoenol pyruvate synthaseCarbohydrate transport and metabolism704
    JCX2D8Upstream of ihfAPutative integration host factor alpha subunit IhfADNA replication, recombination, and repairNA
    9F5nthPutative endonuclease IIIDNA replication, recombination, and repair150
    7D6nthPutative endonuclease IIIDNA replication, recombination, and repair134
    JCX2E12BPSL0175/BPSL1060Putative hypothetical bacteriophage proteinUnknown149
    JCXD3BPSL0175/BPSL1060Putative hypothetical bacteriophage proteinUnknown123

    • ↵a Insertion sites within the capsule region are indicated in Fig. 4b.

    • ↵b NA, not applicable.

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