Construction and characterization of an unmarked aroC deletion mutant of Burkholderia pseudomallei strain A2.

Using a sucrose counter-selection strategy, we have constructed an unmarked aroC deletion mutant of B. pseudomallei strain A2 in order to investigate the possibility of an effective live-attenuated vaccine. The aroC deletion in the resultant mutant, designated A2DeltaaroC, was confirmed by PCR, Southern hybridization and failure of the mutant to grow in a defined medium without aromatic compounds. Compared to the parental wild type strain, A2DeltaaroC was highly attenuated for virulence following intraperitoneal introduction into BALB/c and C57BL/6 mice. BALB/c mice immunized intraperitoneally with A2DeltaaroC were not protected against a challenge dose of 500 cfu (25 x LD50) of the parental strain A2, whereas C57BL/6 mice similarly immunized intraperitoneally with A2DeltaaroC were significantly protected against a challenge dose of 6,000 cfu (20 x LD50).