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

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

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2022.1062682

DOI=10.3389/fcimb.2022.1062682

ISSN=2235-2988

ABSTRACT=<sec><title>Introduction</title><p><italic>Burkholderia pseudomallei</italic>, a soil-dwelling microbe that infects humans and animals is the cause of the fatal disease melioidosis. The molecular mechanisms that underlie <italic>B. pseudomallei’s</italic> versatility to survive within a broad range of environments are still not well defined.</p></sec><sec><title>Methods</title><p>We used the genome-wide screening tool TraDIS (Transposon Directed Insertion-site Sequencing) to identify <italic>B. pseudomallei</italic> 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 <italic>Caenorhabditis</italic> elegans infection model was used to assess genes associated with in vivo <italic>B. pseudomallei</italic> 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.</p></sec><sec><title>Results</title><p>Approximately 500,000 transposon-insertion mutants of <italic>B. pseudomallei</italic> strain R15 were generated. A total of 848,811 unique transposon insertion sites were identified in the <italic>B. pseudomallei</italic> 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 <italic>B. pseudomallei</italic> survival and adaptation in the <italic>C. elegans</italic> intestinal lumen, were identified. When compared to wild type <italic>B. pseudomallei</italic>, a Tn5 mutant of bpsl2988 exhibited reduced survival in the worm intestine, was attenuated in <italic>C. elegans</italic> killing and showed decreased colonization in the organs of infected mice.</p></sec><sec><title>Discussion</title><p>The <italic>B. pseudomallei</italic> conditional essential proteins should provide further insights into the bacteria’s niche adaptation, pathogenesis, and virulence.</p></sec>