Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survivalin vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuationin vivowere identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarkedbpsl2248,tex,rpiR,bpsl1728, andbpss1528deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was testedin vitroandin vivoto confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important toin vivovirulence with roles in different stages ofB. pseudomalleipathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and thetexmutant was capable of providing protective immunity against challenge with wild-typeB. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.
Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei
