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

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.

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

“…3C and D). The attenuation and loss of polysaccharide observed in this mutant seem to correlate with previously published studies (2,33,34).…”

“…3). This confirmed previous data indicating that insertions in wcbB resulted in a loss of capsular polysaccharide (2,33). A summary of transposon insertions identified within the capsule polysaccharide biosynthesis region is shown (Fig.…”

“…A large number of transposon insertions were found in the capsular polysaccharide cluster first described by Reckseidler et al in 2000 (33). The capsular polysaccharide of B. pseudomallei has been shown to be critical for virulence in a number of previous studies (2,33,34).…”

“…The capsular polysaccharide of B. pseudomallei has been shown to be critical for virulence in a number of previous studies (2,33,34). In order to better understand the reason behind the apparent attenuation, the capsular polysaccharide of B. pseudomallei K96243 and a selected panel of transposon derivatives were visualized by immunofluorescence (Fig.…”

“…Currently, no vaccine against the organism exists, and antibiotic treatment is complicated by the fact that B. pseudomallei is intrinsically resistant to many antibiotics including aminoglycosides and the early ␤-lactams (4). A number of virulence determinants including flagella (10), a capsular polysaccharideencoding region (33), and an Inv/Mxi-Spa-like type III secretion system involved in the intracellular behavior of the pathogen (38,39) have been identified in this organism. However, further studies are required to understand this complex infection.…”