Interrogating Genetic Diversity in Mycobacterium abscessus with Transposon-Sequencing

Abstract

Non-tuberculous mycobacteria (NTMs) are free-living species found predominantly in the environment. Of the >200 NTM species identified to date, Mycobacterium abscessus (Mab) is one of the most clinically relevant. Mab is the third most common NTM infection in the United States and is the leading cause of pulmonary infections due to rapidly growing mycobacteria. As the prevalence of Mab infections rise worldwide, treatment options remain poor, with Mab being resistant to many classes of antibiotics. Mab strains are also highly genetically diverse with large accessory genomes. The consequence of this diversity is not well understood, especially as it relates to traits like virulence and antibiotic resistance. Importantly, developing novel antibiotic treatments against Mab requires a better understanding of the essential biological processes in all Mab isolates that can be targeted. In Chapter 1 of my dissertation, I comprehensively review the functional consequence of genetic diversity between clinical isolates. In Chapter 2, we use transposon-sequencing (TnSeq) to identify essential genes in Mab. We further characterize MAB_3167c, which encodes PBP-lipo, a cell-wall enzyme that is essential in Mab and not in Mtb. We find that PBP-lipo localizes to the septum and that repressing PBP-lipo causes Mab cells to elongate and branch. These data suggest that PBP-lipo coordinates peptidoglycan synthesis at the Mab septum and is required for normal cell division. We also identify cell wall enzymes that synergize with PBP-lipo in Mab, but not in M. smegmatis. This data demonstrates that the two organisms have different genetic networks for their cell wall enzymes. Finally, we find that repressing PBP-lipo sensitizes Mab to a range of antibiotics, increasing the therapeutic potential of targeting PBP-lipo. In Chapter 3, we perform TnSeq on 21 Mab clinical isolates to identify genes that are differentially required between strains. We discover gene requirements vary by lineage and strain-specific manners, and show for the first time evolution of gene requirement in Mab. In all, this work establishes the foundation for future studies characterizing essential genes in Mab, which will not only increase our understanding of the basic biology of this understudied pathogen, but also identify new targets for treatment.