Detection of bacterial virulence genes by subtractive hybridization: Identification of capsular polysaccharide of Burkholderia pseudomallei as a major virulence determinant

Burkholderia pseudomallei, the etiologic agent of melioidosis, is responsib le for a broad spectrum of illnesses in humans and animals particularly in Southeast Asia and northern Australia, where it is endemic. Burkholderia th ailandensis 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. pseudomalle i. Screening of the subtraction library revealed A-T-rich DNA sequences uni que to B. pseudomallei, suggesting they may have been acquired by horizonta l transfer. One of the subtraction clones, pDD1015, encoded a protein with homology to a glycosyltransferase from Pseudomonas aeruginosa. This gene wa s insertionally inactivated in wild-type B. pseudomallei to create SR1015. It was determined by enzyme-linked immunosorbent assay and immunoelectron m icroscopy that the inactivated gene was involved in the production of a maj or surface polysaccharide. The 50% lethal dose (LD50) for wild-type B. pseu domallei is <10 CFU; the LD50 for SR1015 was determined to be 3.5 x 10(5) C FU, similar to that of B. thailandensis (6.8 x 10(5) CFU). DNA sequencing o f the region flanking the glycosyltransferase gene revealed open reading fr ames similar to capsular polysaccharide genes in Haemophilus influenzae, Es cherichia coli, and Neisseria meningitidis. In addition, DNA from Burkholde ria mallei and Burkholderia stabilis hybridized to a glycosyltransferase fr agment 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 s tudies has facilitated the identification of an important virulence determi nant in B. pseudomallei.