A mutant of Burkholderia pseudomallei, auxotrophic in the branched chain amino acid biosynthetic pathway, is attenuated and protective in a murine model of melioidosis

Abstract

Using a transposon mutagenesis approach, we have identified a mutant of Burkholderia pseudomallei that is auxotrophic for branched chain amino acids. The transposon was shown to have interrupted the ilvI gene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneally with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-type B. pseudomallei.

FIG. 1.

Growth of B. pseudomallei mutant 2D2 and strain 576 in defined growth media. M9 growth medium was prepared as previously described (36) and was supplemented with leucine, isoleucine, and valine (40 μg/ml) where appropriate. The results represent the mean of three experiments. OD₆₀₀, optical density at 600 nm.

FIG. 2.

Clearance of B. pseudomallei mutant 2D2 (▪) or strain 576 (♦) from animal tissue. Groups of five female BALB/c mice were inoculated with 10⁴ CFU by the intraperitoneal route of infection. At time points throughout the duration of the experiment mice were killed and liver (A), spleen (B), kidney (C), and lung (D) were harvested. The number of B. pseudomallei organisms present in each organ was calculated (error bars represent mean bacterial counts from three individual animals).