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

Abstract

ABSTRACT 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 .