title: Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei.
abstract: 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 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 with various degrees of attenuation in vivo were identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarked bpsl2248, tex, rpiR, bpsl1728, and bpss1528 deletion mutants were constructed from 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.
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Madeleine G
authors.initials: MG
authors.lastname: Moule
authors.affiliation: Department of Infection and Immunity, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Natasha
authors.initials: N
authors.lastname: Spink
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Sam
authors.initials: S
authors.lastname: Willcocks
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Jiali
authors.initials: J
authors.lastname: Lim
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: José Afonso
authors.initials: JA
authors.lastname: Guerra-Assunção
authors.affiliation: Department of Infection and Immunity, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Felipe
authors.initials: F
authors.lastname: Cia
authors.affiliation: School of Biosciences, University of Exeter, Devon, United Kingdom.
authors.forename: Olivia L
authors.initials: OL
authors.lastname: Champion
authors.affiliation: School of Biosciences, University of Exeter, Devon, United Kingdom.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Senior
authors.affiliation: Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire, United Kingdom.
authors.forename: Helen S
authors.initials: HS
authors.lastname: Atkins
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Taane
authors.initials: T
authors.lastname: Clark
authors.affiliation: Department of Infection and Immunity, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Bancroft
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Jon
authors.initials: J
authors.lastname: Cuccui
authors.affiliation: Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom bwren@lshtm.ac.uk.
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016957:Burkholderia pseudomallei / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D005260:Female; D006801:Humans; D008554:Melioidosis / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors