Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium
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Abstract
The cell envelope of Gram-negative bacteria belonging to the Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals. Here, we present the results of a genome-wide screen for cell envelope-associated resistance and susceptibility determinants in a Burkholderia cenocepacia clinical isolate. For this purpose, we construct a high-density, randomly-barcoded transposon mutant library and expose it to 19 cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profile over a hundred functional associations and identify mediators of antibiotic susceptibility in the Bcc cell envelope. We reveal connections between β-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism. The synergy of the β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. In comparison with ceftazidime, avibactam more strongly potentiates the activity of aztreonam and meropenem in a panel of Bcc clinical isolates. Finally, we characterize in Bcc the iron and receptor-dependent activity of the siderophore-cephalosporin antibiotic, cefiderocol. Our work has implications for antibiotic target prioritization, and for using additional combinations of β-lactam/β-lactamase inhibitors that can extend the utility of current antibacterial therapies.
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General comments:
1-Overall a really important paper! Thank you! We need to protect our current antibiotic arsenal as well as develop new antibiotics - this paper provides insight into how to do both!
2-Specifically, beta-lactams are such an important class of antibiotics and we should strive to do our best to preserve their potency and also find new determinants that could help us re-sensitize already resistant bacteria.
3-The authors have done a lot of work in this paper! Really great job! First constructing the transposon library, testing it across many different antibiotics, then validating many of the identified genes and pathways.
4-I love that the authors focused on the cell envelope! It’s an orthogonal cell structure to human cells, it’s easily accessible, and I really think there is still so much we don’t know about how …
General comments:
1-Overall a really important paper! Thank you! We need to protect our current antibiotic arsenal as well as develop new antibiotics - this paper provides insight into how to do both!
2-Specifically, beta-lactams are such an important class of antibiotics and we should strive to do our best to preserve their potency and also find new determinants that could help us re-sensitize already resistant bacteria.
3-The authors have done a lot of work in this paper! Really great job! First constructing the transposon library, testing it across many different antibiotics, then validating many of the identified genes and pathways.
4-I love that the authors focused on the cell envelope! It’s an orthogonal cell structure to human cells, it’s easily accessible, and I really think there is still so much we don’t know about how antibiotics target the cell envelope and lead to lysis or stasis!
5-I also love the barcoded transposon approach! It provides a wealth of information and it can be applied across different species but the utility got so much higher once the barcodes were introduced! Kudos for doing that!
6-Overall, I think this paper also shines light on the large differences in cell wall physiology even in the more closely-related Gram-negative bacteria. I hope people branch out more and venture frequently outside of the E. coli world.
Specific comments:
1-Is there biochemical evidence indicating that beta-lactam antibiotics have the same targets in B. cenocepacia as they do in other bacteria (E. coli)?
1-Do you think it’s worth conducting your screen in other conditions besides LB media? I am wondering how much of the findings in LB media translate to antibiotic resistance in the real world?
2-Is there any way to adjust your barcoded transposon approach so that it can be used to probe the role of essential genes in antibiotic resistance? Or do you think you would need a completely different strategy for that? Perhaps a high-throughput CRISPRi approach that would produce only a knockdown in gene expression?
3-Your finding that cycloserine could have an effect on D-glutamate synthesizing enzymes is really exciting! I encourage you or somebody to follow up on that! It would be cool if this leads to developing cycloserine analogs that inhibit D-glutamate production.
4-Similarly to point 3, I was really intrigued by the differences in UndP metabolism in B. cenocepacia compared to E. coli. More specifically, is it known how many UndPP phosphatases there are in B. cenocepacia? I also love your discussion on this point! It’s so cool when people venture outside of the established model organisms and find that conserved physiological processes actually show tremendous variability!
Questions:
1-Is it known if the outer membrane in Burkholderia is responsible for handling any of the turgor pressure? Typically the assumption is that peptidoglycan is the major structure that bears the pressure but we know now that membranes play a role as well (at least in some bacteria). I am wondering if people have studied that in Burkholderia because their outer membrane has distinct properties from other Gram-negatives.
2-Out in nature, do you know if Burkholderia is antagonized by any contact-dependent mechanisms? I’m thinking any bacteria with a Type VI secretion system like Pseudomonas aeruginosa for example. I think it could be cool to use your barcoded transposon approach to look for susceptibility determinants towards Type VI secreted toxins that target the cell wall!
3-I wonder if there is a way to summarize your findings in a database or a resources website? Is there a Burkholderia database that harbors antibiotic resistance information? I think having a publication is great but the utility of your work would skyrocket if the general public, policy makers, and medical doctors could access your findings more quickly and directly.
4-Do you think some of the phenotypes you have measured could be due to polar effects caused by an overall operon disruption? It could be worth following up with some of your knockout strains for genes in operons and measuring the expression of neighboring genes in that same operon.
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