Genome-wide transposon mutagenesis analysis of Burkholderia pseudomallei reveals essential genes for in vitro and in vivo survival

Yee-Chin Wong, Raeece Naeem, Moataz Abd El Ghany, Chee-Choong Hoh, Arnab Pain, Sheila Nathan

Research output: Contribution to journalArticlepeer-review

Abstract

Introduction: Burkholderia pseudomallei, a soil-dwelling microbe that infects humans and animals is the cause of the fatal disease melioidosis. The molecular mechanisms that underlie B. pseudomallei’s versatility to survive within a broad range of environments are still not well defined. Methods: We used the genome-wide screening tool TraDIS (Transposon Directed Insertion-site Sequencing) to identify B. pseudomallei essential genes. Transposon-flanking regions were sequenced and gene essentiality was assessed based on the frequency of transposon insertions within each gene. Transposon mutants were grown in LB and M9 minimal medium to determine conditionally essential genes required for growth under laboratory conditions. The Caenorhabditis elegans infection model was used to assess genes associated with in vivo B. pseudomallei survival. Transposon mutants were fed to the worms, recovered from worm intestines, and sequenced. Two selected mutants were constructed and evaluated for the bacteria’s ability to survive and proliferate in the nematode intestinal lumen. Results: Approximately 500,000 transposon-insertion mutants of B. pseudomallei strain R15 were generated. A total of 848,811 unique transposon insertion sites were identified in the B. pseudomallei R15 genome and 492 genes carrying low insertion frequencies were predicted to be essential. A total of 96 genes specifically required to support growth under nutrient-depleted conditions were identified. Genes most likely to be involved in B. pseudomallei survival and adaptation in the C. elegans intestinal lumen, were identified. When compared to wild type B. pseudomallei, a Tn5 mutant of bpsl2988 exhibited reduced survival in the worm intestine, was attenuated in C. elegans killing and showed decreased colonization in the organs of infected mice. Discussion: The B. pseudomallei conditional essential proteins should provide further insights into the bacteria’s niche adaptation, pathogenesis, and virulence.
Original languageEnglish (US)
JournalFrontiers in cellular and infection microbiology
Volume12
DOIs
StatePublished - Dec 23 2022

Bibliographical note

KAUST Repository Item: Exported on 2023-01-12
Acknowledged KAUST grant number(s): BAS/1/1020-01-01
Acknowledgements: This study was supported by the grants 06-05-16-MB003 from the Ministry of Science, Technology and Innovation Malaysia and DIP-2015-022 from Universiti Kebangsaan Malaysia awarded to SN. YW was supported by a MyBrain15 Scholarship from the Ministry of Higher Education Malaysia. Part of this work was conducted in KAUST, funded by the faculty baseline fund BAS/1/1020-01-01 from KAUST to AP.

ASJC Scopus subject areas

  • Microbiology (medical)
  • Infectious Diseases
  • General Medicine
  • Immunology
  • Microbiology

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