High-density transposon mutagenesis in Mycobacterium abscessus identifies an essential penicillin-binding lipo-protein (PBP-lipo) involved in septal peptidoglycan synthesis and antibiotic sensitivity

Abstract

Mycobacterium abscessus (Mab) is a rapidly growing non-tuberculous mycobacterium (NTM) that causes a wide range of infections. Treatment of Mab infections is difficult because the bacterium is intrinsically resistant to many classes of antibiotics. Developing new and effective treatments against Mab requires a better understanding of the unique vulnerabilities that can be targeted for future drug development. To achieve this, we identified essential genes in Mab by conducting transposon-sequencing (TnSeq) on the reference Mab 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 Mab, we further characterized MAB_3167c, a predicted penicillin-binding-lipoprotein (PBP-lipo) that is essential in Mab and non-essential in M. tuberculosis (Mtb). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Knockdown of Mab PBP-lipo causes cells to elongate, develop ectopic branches, and form multiple septa. Dual knockdown of PBP-lipo with PBPs, PbpB, DacB1, and a carboxypeptidase, MAB_0519 lead to synergistic growth arrest. In contrast, these genetic interactions were absent in the Mtb model organism, M. smegmatis, 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 Mab clinical isolates to several classes of antibiotics, including the beta-lactams, ampicillin and amoxicillin by greater than 128-fold. Altogether, this study presents PBP-lipo as a key enzyme to study Mab specific processes in cell wall synthesis and importantly positions PBP-lipo as an attractive drug target to treat Mab infections.

Transposon-sequencing across multiple Mycobacterium abscessus isolates reveals significant functional genomic diversity among strains

“…Conversely, genes classified as ‘growth advantage’ cause a fitness benefit relative to wildtype when they are disrupted, resulting in increased insertion counts. The essential genes of the reference strain, ATCC 19977 have been previously reported (25, 27). We compared our predicted essential or growth-defect genes for ATCC 19977 to those in Rifat et al (25), and found an overlap of 393 genes, showing high concordance between these two studies of Mab essentials (Supplemental Figure 2A).…”

“…Whole Genome Sequencing of Mab Clinical Isolates: Genomic DNA from late-log phase cultures were extracted via a bead-beating and phenol-chloroform protocol described in Akusobi et al (27), using an Illumina Rapid HiSeq 2500 instrument with 125+125 bp paired-end reads.…”

“…Clinical isolates were streaked from patient stabs, single colonies were picked, cultured, and frozen for future use. Strains with transformed CRISPRi/Cas9 vectors to knockdown central carbon metabolism genes were built as described in (27). All strains were grown shaking at 37°C in liquid 7H9 media consisting of Middlebrook 7H9 salts with 0.2% glycerol, 0.85 g/L NaCl, and OADC (oleic acid, 5 g/L albumin, 2 g/L dextrose, 0.003 g/L catalase), and 0.05% Tween80.…”

“…High-density transposon libraries of selected Mab clinical isolates were generated using the ϕMycoMarT7 phage as described in Akusobi et al (27). Libraries were generated in biological triplicate for 15 clinical isolates, while single replicate libraries were generated for 6 isolates (see Supplemental Table 4).…”

“…For Mab, the majority of TnSeq studies published to date have used the lab-adapted reference strain, ATCC 19977 (25)(26)(27). One TnSeq study was conducted on a massiliense clinical isolate, however the transposon library was not dense enough to quantify genetic requirement (28).…”