Characterization of new virulence factors involved in the intracellular growth and survival of Burkholderia pseudomallei.
Moule, MG; Spink, N; Willcocks, S; Lim, J; Guerra-Assunção, JA; Cia, F; Champion, O; Senior, N; Atkins, HS; Clark, T; Bancroft, GJ; Cuccui, J; Wren, BW; (2015) Characterization of new virulence factors involved in the intracellular growth and survival of Burkholderia pseudomallei. Infection and immunity. ISSN 0019-9567 DOI: 10.1128/IAI.01102-15
Burkholderia pseudomallei, the causative agent of melioidosis, has a complex and poorly understood extracellular and intracellular lifestyle. We used transposon 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 survival in 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 were identified with varying degrees of attenuation in vivo. Five of the newly identified genes were chosen for further characterization and clean, unmarked deletion mutants of bpsl2248, tex, rpiR, bpsl1728 and bpss1528 were constructed in the wild-type strain K96243. Each of these mutants was tested in vitro and in vivo to confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important to in vivo virulence with roles in different stages of B. pseudomallei pathogenesis including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating and the tex mutant was capable of providing protective immunity against challenge with wild-type B. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.
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