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

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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 Mycobacterium tuberculosis ( Mtb ). We found that PBP-lipo primarily localizes to the subpolar region and later to the septum as cells prepare to divide. Depletion of Mab 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 Mtb model organism, Mycobacterium 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 β-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.

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  1. Evaluation Summary:

    This study reports the results of a transposon inactivation screen to identify essential genes in Mycobacterium abscessus. The authors investigate one hit, the gene encoding the class B penicillin-binding protein, PBP-lipo. They confirm that the PBP-lipo gene is essential despite the presence of a homologous gene and that PBP-lipo is present in other mycobacteria, but not essential in these. They further characterize the consequences of PBP-lipo gene depletion in M. abscessus and demonstrate that the gene product is required for maintaining cell morphology, whilst also participating in a network with other cell wall enzymes. The manuscript will be of interest for researchers working on fundamental aspects of cell wall synthesis in mycobacteria. It may also be relevant for researchers who aim to specifically target M. abscessus using new drugs or drug combinations.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    The authors identified the gene encoding PBP-lipo by a Tn-Seq analysis of the Mycobacterium abscessus genome seeking genes required for viability. PBP-lipo essentiality was confirmed by CRSPRi knockdown (KD) studies and the mutant/KD phenotypes (cell shape and cytokinetic defects) observed by microscopy. The show that PBP-lipo localizes to the division plane and interacts genetically (by conditional CRISPRi partial KD) with other cell wall biosynthesis proteins that are known to act at the division plane. they also find PBP-lipo is also required to protect against antibiotics that target the cell wall machinery and also some antibiotics that act in the cytoplasm, therefore indicating that PBP-lipo KD mutants suffer from a permeability defect which they could recapitulate by calcein staining.

    Importantly, they find that PBP-lipo is important in Mycobacterium abscessus (including clinical isolates) but not in Mycobacterium smegmatis and Mycobacterium tuberculosis lab strains. The authors show that Mycobacterium smegmatis PBP-lipo can, however, compensate for the loss of PBP-lipo in Mycobacterium abscessus, showing that the ortholog of Mycobacterium smegmatis has not lost its function, but that the idiosyncracies of the cell wall biosynthesis machineries and their interaction network in various mycobacteria differs. These findings set the stage for (combinatorial) beta-lactam based treatments of Mycobacterium abscessus infections.

    The work is exceptionally well done, comprehensive, solid and clearly presented.

  3. Reviewer #2 (Public Review):

    In my view the authors used suitable methodologies to address important research questions in mycobacteria: What are the essential genes of Mab and why is one of these, the PBP-lipo gene essential in Mab despite the presence of a homologous gene. They also revealed differences in PBP requirements in different mycobacterial species. In my view, this is an excellent manuscript demonstrating the importance of a new PBP for Mab growth, morphology and sensitivity to antibiotics. However, I noticed that a recent publication has already reported the essential genes in Mab using a similar methodology. It is also fair to say that it is not a novelty that bacteria have different sets of PBPs and some PBPs are essential in one species but not in others, sometimes even closely related species. This wider aspect beyond mycobacteria is not well discussed in the manuscript. Nevertheless, I believe that the manuscripts provides an important step forward in understanding cell wall biogenesis in different mycobacteria, revealing an important cell wall enzyme in Mycobacterium abscessus that could be targeted by drugs against this pathogen.

  4. Reviewer #3 (Public Review):

    In this study, Akusobi et al., compare the inventory of Tn-seq-derived essential genes in M. abscessus, to those in M. tuberculosis and M. smegmatis and identify a set of genes that are 'uniquely essential' to Mab. A subset of these 'uniquely essential' genes are validated using CRISPRi knockdown. They select PBP-lipo, a putative Class B penicillin-binding lipoprotein, for further analyses. Using a combination of neat genetic and cell biology experiments they characterize the possible role of PBP-lipo in regulating growth and development of Mab. PBP-lipo localizes to the putative division site and depletion of PBP-lipo produces branched filamentous cells with decreased viability. Interestingly, expression of Msm PBP-lipo could reverse the developmental defects of PBP-lipo depleted Mab cells suggesting it may not be the property of the protein but its interacting network that might be unique in Mab. Genetic interaction studies using other annotated PBPs in Mab suggests that PBP-lipo may function in tandem with pbpB, dacB1 and MAB_0519, of which DacB1 and PbpB may localize to the same sub-cellular position as PBP-lipo. These genetic interactions are not seen in Msm and PBP-lipo does not have a synthetic lethal partner in Msm. Strikingly, PBP-lipo depletion renders both laboratory strain and clinical isolates of Mab susceptible to a subset of antibiotics.

    This is an elegantly carried out study that demonstrates the unique role of PBP-lipo in maintaining envelope integrity of Mab. This exciting and timely finding not only opens up the possibility of using PBP-lipo as a putative drug target in treating Mab infections, it further goes onto set the stage for further in-depth analyses of species-specific developmental processes in Mab.