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Title:	High-throughput sequencing of transposon mutants to identify essential genes of Burkholderia pseudomallei and Burkholderia cenocepacia
Authors:	Wong Yee Chin (P58662)
Supervisor:	Sheila Nathan, Prof. Dr.
Keywords:	Transposon mutants Burkholderia pseudomallei Burkholderia cenocepacia Genes Dissertations, Academic -- Malaysia
Issue Date:	28-Sep-2016
Description:	Essential genes are the minimum set of genes required to sustain bacterial viability. Studies on gene essentiality are aimed at unravelling the vital cellular functions of a pathogen whilst contributing to the understanding of the mode of pathogenesis. In this study, TraDIS (Transposon Directed Insertion-site Sequencing) was utilised as a genome-wide screening tool to facilitate the identification of essential genes of two human-pathogenic Burkholderia species, namely Burkholderia pseudomallei, the causative agent of the fatal disease melioidosis, and Burkholderia cenocepacia, an emerging threat to cystic fibrosis patients. Using Tn5-based mutagenesis, approximately 500,000 transposon-insertion mutants each of both B. pseudomallei strain R15 and B. cenocepacia strain J2315 were generated. To identify unique transposon insertion sites, transposon flanking regions were sequenced on the Illumina next-generation sequencing platform. Gene essentiality was then assessed based on the frequency of transposon insertions within each gene. Out of 54.82 million reads mapped to the B. pseudomallei R15 genome, 848,811 unique transposon insertion sites were identified. Of these, 492 genes with low insertion frequencies were predicted to be essential. As for B. cenocepacia J2315, 422,585 unique insertion sites were detected, with 383 genes predicted as essential genes. To determine the sets of conditionally essential genes required for in vitro growth, transposon mutants (input pool) were grown in LB and M9 minimal medium. Genes specifically required for B. pseudomallei (96) and B. cenocepacia (439) growth under nutrient-depleted conditions were identified. To determine the set of genes required for an active infection of the host, genes associated with B. pseudomallei and B. cenocepcia survival in vivo, were assessed in the Caenorhabditis elegans infection model. Transposon mutants (input pool) were used to infect worms and at different times post-infection, mutants were recovered from worm intestines (output pools). By using TraDIS to detect loss of mutants, panels of genes were predicted to be most likely involved in B. pseudomallei and B. cenocepacia survival and adaptation in the C. elegans intestinal lumen. To confirm the TraDIS prediction, deletion mutants of selected genes were constructed and the ability of the mutants to survive and proliferate in the C. elegans intestinal lumen was assessed. The roles of these genes in relation to other virulence-associated phenotypes were investigated. Of particular interest, when compared to wild type B. pseudomallei, a Tn5 mutant of bpsl2988 (encoding a sugar kinase) exhibited reduced survival in the worm intestinal lumen and was attenuated in C. elegans killing. In addition, the bpsl2988 mutant showed decreased colonization in the organs of infected mice, particularly in the lung. Deletion of a histone-like nucleoid structuring (H-NS) protein encoded by bcal0154 was shown to impact the colonization ability and virulence of B. cenocepacia in C. elegans, as well as its motility and biofilm formation in vitro. Further functional characterization of these genes should provide insights into Burkholderia species variation in relation to mode of infection, niche adaptation, pathogenesis and also virulence.,Certification of Master's/Doctoral Thesis" is not available
Pages:	185
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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