Transposon mutagenesis in <i>Mycobacterium abscessus</i> identifies an essential penicillin-binding protein involved in septal peptidoglycan synthesis and antibiotic sensitivity.

Abstract

<i>Mycobacterium abscessus</i> (<i>Mab</i>) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of <i>Mab</i> infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against <i>Mab</i> requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in <i>Mab</i> by conducting transposon sequencing (TnSeq) on the reference <i>Mab</i> strain ATCC 19977. We generated ~51,000 unique transposon mutants and used this high-density library to identify 362 essential genes for in vitro growth. To investigate species-specific vulnerabilities in <i>Mab</i>, we further characterized <i>MAB_3167c</i>, a predicted penicillin-binding protein and hypothetical lipoprotein (PBP-lipo) that is essential in <i>Mab</i> and non-essential in <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of <i>Mab</i> PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Knockdown of PBP-lipo along with PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the <i>Mtb</i> model organism, <i>Mycobacterium smegmatis</i>, indicating that the PBP-lipo homologs in the two species exist in distinct genetic networks. Finally, repressing PBP-lipo sensitized the reference strain and 11 <i>Mab</i> clinical isolates to several classes of antibiotics, including the β-lactams, ampicillin, and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study <i>Mab</i>-specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat <i>Mab</i> infections.