Identification of a Burkholderia mallei polysaccharide gene duster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant

Little is known about the virulence factors of Burkholderia mallei, the eti ologic agent of glanders, We employed subtractive hybridization to identify genetic determinants present in B. mallei but not in Burkholderia thailand ensis, a non-pathogenic soil microbe. Three subtractive hybridization produ cts were mapped to a genetic locus encoding proteins involved in the biosyn thesis, export and translocation of a capsular polysaccharide. We identifie d an insertion sequence (IS407A) at one end of the capsule gene cluster and demonstrated that it was functional in B. mallei. Mutations were introduce d in the B. mallei capsular gene cluster and the corresponding mutants were examined for their reactivity with antibodies raised against Burkholderia pseudomallei surface polysaccharides by immunoblotting and ELISA. Immunogol d electron microscopy demonstrated the presence of a capsule on the surface of B. mallei ATCC 23344 (parental strain) but not on B. mallei DD3008 (cap sule mutant) or B. thailandensis. Surprisingly, B. thailandensis also harbo ured a portion of the capsule gene cluster. ATCC 23344 was highly virulent in hamsters and mice, but DD3008 was avirulent in both animal models. The r esults presented here demonstrate that the capsular polysaccharide of B. ma llei is required for production of disease in two animal models of glanders infection and is a major virulence factor.