Detection of Bacterial Virulence Genes by Subtractive Hybridization: Identification of Capsular Polysaccharide of <i>Burkholderia pseudomallei</i> as a Major Virulence Determinant

• Shauna L. Reckseidler

  <!--label omitted: 1-->Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada T2N 4N1,1 and

• David DeShazer

  <!--label omitted: 2-->Bacteriology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 217022

• Pamela A. Sokol

  <!--label omitted: 1-->Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada T2N 4N1,1 and

• Donald E. Woods

  <!--label omitted: 1-->Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada T2N 4N1,1 and

• V. J. DiRita

  editor

抄録

Burkholderia pseudomallei , the etiologic agent of melioidosis, is responsible for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic. Burkholderia thailandensis is a nonpathogenic environmental organism closely related to B. pseudomallei . Subtractive hybridization was carried out between these two species to identify genes encoding virulence determinants in B. pseudomallei . Screening of the subtraction library revealed A-T-rich DNA sequences unique to B. pseudomallei , suggesting they may have been acquired by horizontal transfer. One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from Pseudomonas aeruginosa . This gene was insertionally inactivated in wild-type B. pseudomallei to create SR1015. It was determined by enzyme-linked immunosorbent assay and immunoelectron microscopy that the inactivated gene was involved in the production of a major surface polysaccharide. The 50% lethal dose (LD 50 ) for wild-type B. pseudomallei is <10 CFU; the LD 50 for SR1015 was determined to be 3.5 × 10 5 CFU, similar to that of B. thailandensis (6.8 × 10 5 CFU). DNA sequencing of the region flanking the glycosyltransferase gene revealed open reading frames similar to capsular polysaccharide genes in Haemophilus influenzae , Escherichia coli , and Neisseria meningitidis . In addition, DNA from Burkholderia mallei and Burkholderia stabilis hybridized to a glycosyltransferase fragment probe, and a capsular structure was identified on the surface of B. stabilis via immunoelectron microscopy. Thus, the combination of PCR-based subtractive hybridization, insertional inactivation, and animal virulence studies has facilitated the identification of an important virulence determinant in B. pseudomallei .

収録刊行物

• Infection and Immunity

  Infection and Immunity 69 (1), 34-44, 2001-01

  American Society for Microbiology

キーワード

• Infectious Diseases
• Immunology
• Microbiology
• Parasitology

詳細情報

• CRID: 1361137045314341376
• NII論文ID: 30020828869
• NII書誌ID: AA00673732
• DOI: 10.1128/iai.69.1.34-44.2001
• ISSN: 10985522
• ISSN: 00199567
• Web Site: https://journals.asm.org/doi/pdf/10.1128/IAI.69.1.34-44.2001
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