Genome-Wide Essentiality Analysis of
            <i>Mycobacterium abscessus</i>
            by Saturated Transposon Mutagenesis and Deep Sequencing

Rifat, Dalin; Chen, Liang; Kreiswirth, Barry N; Nuermberger, Eric

doi:10.1128/mbio.01049-21

Cited by 43 publications

(52 citation statements)

References 104 publications

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“…We found that MAB_3167c lacked transposon insertions across all three replicate libraries (Figure 3A). This finding corroborates data from previously generated Mab transposon libraries that show lack of insertions in MAB_3167c (10, 12). Comparing the essentiality of PBP enzymes in Mab with their homologs in Mtb revealed that MAB_3167c is unique in being the sole PBP whose essentiality differs between the two species.…”

Section: Resultssupporting

confidence: 92%

“…Overall, the newly generated Mab transposon libraries can be used to determine genes required for growth in a variety of conditions including infection models, biofilms, and antibiotic stress. Both Rifat et al’s and our TnSeq data identified PBP-lipo as the sole PBP that is essential in Mab and non-essential in Mtb (12). Using CRISPRi, we demonstrated the necessity of PBP-lipo expression for Mab to maintain normal morphology and to divide properly.…”

Section: Discussionmentioning

confidence: 56%

“…While both Mab TnSeq screens provided tools and important insights into Mab biology, neither utilized a mutant library with high enough density to comprehensively identify essential genes. This changed with the recent publication from Rifat et al , who generated robust high-density transposon libraries of Mab and identified a comprehensive list of genetic elements essential for in vitro growth (12). Identification of these essential genes serves as an excellent starting point to classify attractive targets for drug discovery.…”

Section: Introductionmentioning

confidence: 99%

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

Akusobi

et al. 2021

Preprint

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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 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.

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Section: Resultssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 56%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Akusobi

et al. 2021

Preprint

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“…The predictive power of our model on the M. abscessus data set ( R 2 of 0.504) is slightly higher than, but about the same as, the M. tuberculosis test set. We ran the same analysis on a recently published TnSeq data set from an independent library of M. abscessus ATCC 19977 ( 16 ) as well and observed very similar heatmaps and predictive power ( R 2 of 0.477). This shows we can explain ∼50% of the variance in Himar1 insertion counts in this organism based on the nucleotide biases.…”

Section: Resultsmentioning

confidence: 84%

Modeling Site-Specific Nucleotide Biases Affecting Himar1 Transposon Insertion Frequencies in TnSeq Data Sets

et al. 2021

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When using the Himar1 transposon to create transposon insertion mutant libraries, it is known that the transposon is restricted to insertions at TA dinucleotide sites throughout the genome, and the absence of insertions is used to infer which genes are essential (or conditionally essential) in a bacterial organism. It is widely assumed that insertions in nonessential regions are otherwise random, and this assumption is used as the basis of several methods for statistical analysis of TnSeq data.

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“…Transposon insertion site mapping, using methods such as Tn‐seq or TraDIS, has been widely applied to study essentiality and fitness impact of genes in different organisms (Goodall et al, 2018; Rifat et al, 2021). This technique has also been extended to investigate synthetic interactions between genes via comparative analyses of transposon insertion site profiles in different strain backgrounds (Truong et al, 2020; Winkle et al, 2021).…”

Section: Resultsmentioning

confidence: 99%

Genetic interaction mapping highlights key roles of the Tol‐Pal complex

Tan

Chng

2022

Molecular Microbiology

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The conserved Tol‐Pal trans‐envelope complex is important for outer membrane (OM) stability and cell division in Gram‐negative bacteria. It is proposed to mediate OM constriction during cell division via cell wall tethering. Yet, recent studies suggest the complex has additional roles in OM lipid homeostasis and septal wall separation. How Tol‐Pal facilitates all these processes is unclear. To gain insights into its function(s), we applied transposon‐insertion sequencing, and report here a detailed network of genetic interactions with the tol‐pal locus in Escherichia coli. We found one positive and > 20 negative strong interactions based on fitness. Disrupting osmoregulated‐periplasmic glucan biosynthesis restores fitness and OM barrier function, but not proper division, in tol‐pal mutants. In contrast, deleting genes involved in OM homeostasis and cell wall remodeling causes synthetic growth defects in strains lacking Tol‐Pal, especially exacerbating OM barrier and/or division phenotypes. Notably, the ΔtolA mutant having additional defects in OM protein assembly (ΔbamB) exhibited severe division phenotypes, even when single mutants divided normally; this highlights the possibility for OM phenotypes to indirectly impact cell division. Overall, our work underscores the intricate nature of Tol‐Pal function, and reinforces its key roles in cell wall‐OM tethering, cell wall remodeling, and in particular, OM homeostasis.

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Genome-Wide Essentiality Analysis of Mycobacterium abscessus by Saturated Transposon Mutagenesis and Deep Sequencing

Cited by 43 publications

References 104 publications

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

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

Modeling Site-Specific Nucleotide Biases Affecting Himar1 Transposon Insertion Frequencies in TnSeq Data Sets

Genetic interaction mapping highlights key roles of the Tol‐Pal complex

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