Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products
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Evaluation Summary:
This study addresses a major missing element in the understanding of how bacteria grow their cell wall and the role of lytic transglycosylases in this process. It had been previously assumed these enzymes cut glycan strands to make room for the insertion of new glycans. However, results presented in this manuscript demonstrate these enzymes have a very different, yet essential role in degrading uncrosslinked glycan strands in the periplasm. The authors further demonstrate that in the absence of lytic transglycosylases, cells undergo periplasmic stress due a toxic accumulation of these "free strands" in the periplasm. The work will be of interest to those in the bacterial growth and division field.
(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. Reviewer #2 agreed to share their name with the authors.)
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Abstract
The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover – or autolysis – of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditional LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and, contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.
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Author Response:
Reviewer #3 (Public Review):
In this manuscript the authors make several conclusions, according to the abstract:
1 - LTG activity is essential by contributing to a process independent of PG recycling.
2 - LTGs are important because of their catalytic activity rather than because of a protein-protein interaction.
3 - LTG mutants are hypersusceptible to production of periplasmic polymers.
4 - LTGs prevent toxic periplasmic crowding and their function is temporally separate from PG synthesis.
The authors perform a series of genetic experiments that lead to their conclusions. Their first conclusion is well supported by data showing that a PG recycling mutant does not have the same defects as their LTG mutant.
Their second conclusion needs more justification/explanation. They show a catalytic mutant of RlpA is …
Author Response:
Reviewer #3 (Public Review):
In this manuscript the authors make several conclusions, according to the abstract:
1 - LTG activity is essential by contributing to a process independent of PG recycling.
2 - LTGs are important because of their catalytic activity rather than because of a protein-protein interaction.
3 - LTG mutants are hypersusceptible to production of periplasmic polymers.
4 - LTGs prevent toxic periplasmic crowding and their function is temporally separate from PG synthesis.
The authors perform a series of genetic experiments that lead to their conclusions. Their first conclusion is well supported by data showing that a PG recycling mutant does not have the same defects as their LTG mutant.
Their second conclusion needs more justification/explanation. They show a catalytic mutant of RlpA is unable to sustain growth as the only LTG in the cell. However, I am confused by their wording around RlpA in general. In the text they note that their delta_7 mutant, which encodes RlpA, 'has no highly active LTGs' (lines 130-131). Does that imply that RlpA is not an LTG? In the discussion they note that E.coli RlpA has no LTG activity. Is this enzyme known to have LTG activity in V.cholerae? One important control would be to show that the catalytically inactive protein is stable (i.e. that the defect is not due to protein misfolding). This could be supported by looking at protein stability via Western or even quantifying the fluorescence data in Figure S3b.
Alignment of VcRlpA with P. aeruginosa RlpA, which has been demonstrated in vivo and in vitro to be an active LTG, suggests VcRlpA retains the active site residues required for PG cleavage. This, as well as the inability of a VcRlpA^D145A mutant (based on the alignment with catalytically inactive EcRlpA) to rescue native RlpA depletion from the ∆LTG mutants suggests that VcRlpA is an active LTG and that this activity is required in the absence of all other annotated V. cholerae LTGs. We agree that “no highly active LTGs” is confusing and we have changed the text to simply describe the ∆7 LTG mutant as being significantly depleted in LTG activity as measured by anhMurNAc abundance in the sacculus. Lastly, we have conducted Western Blots demonstrating in the revised manuscript that our catalytic site mutant is indeed produced and stable (Figure S3).
Their third conclusion also needs more support. The authors do a series of experiments showing that delta7 is more susceptible to SacB. What are the data that show sacB produces large polysaccharides molecules in the periplasm rather than (or in addition to) the cytoplasm? This would be important to show as these data are the main test of the authors model.
In native B. subtilis as well as in E. coli, SacB has a canonical Sec signal peptide which is annotated as being cleaved after residue Ala29 (Uniprot G3CAF6_BACIU) to be released extracellularly. A reference (Pereira, et al, 2001) has been added in support of SacB functioning extracellularly and not in the cytoplasm of its native host, B. subtilis.
The authors have other data that all argue for their model that LTG deficient strains have an excess of periplasmic crowding. The suppressor of delta_opgH is intriguing, but does not restore the morphological defects in delta_7, suggesting that the increase in length during prolonged growth may not be caused by periplasmic crowding, or at least is not alleviated by deletion of OpgH. What then does the deletion of OpgH suppress? Here, I was confused by the experiments in low salt. The authors write that the cells lyse (line 222) but this is not shown anywhere. Growing the cells continually in low salt may not be the hypoosmotic challenge the authors presume. A challenge typically implies an acute change in osmolarity, rather than a prolonged exposure, which may allow cells to adapt.
We do not fully understand the role of OpgH, but here is our working model: LTGs have at least two essential functions – 1) PG release and 2) mitigating periplasmic crowding, either or both of which can become more important based on osmotic conditions. Since MltG seems to be the main PG release factor (at least based on E. coli), which can be partially supplanted by collective action of other LTGs, the ∆7 suffers from both PG release defects and periplasmic crowding defects, perhaps more so in an osmotically challenging low salt medium. The evidence for lysis is that at high inoculum (10^-2) the ∆7 LTG mutant does grow for a short time, but then we observe a drop in OD_600, indicative of lysis. According to our model, ∆6, on the other hand, which still has MltG, likely suffers only (or mostly) from a periplasmic crowding defect. Deleting periplasmic glucans only mitigates periplasmic crowding (and probably only partially), which does not help the more defective ∆7, which additionally suffers from lack of the postulated second activity.
The reviewers raise an interesting point regarding the word “challenge”. We indeed specifically make the point that this is not an acute challenge, but rather accumulating damage during prolonged growth, even in salt-free LB. We have thus removed the word “challenge” from the revised manuscript. Importantly, we only use the ∆opgH suppression phenotype as one of many puzzle pieces for our conclusion. The key assay is the direct demonstration of periplasmic soluble PG strands accumulating in both WT and, to a higher degree, the ∆6 LTG mutant (Fig. 6).
I was also highly confused by the antibiotic + BADA staining experiments. Do the authors stain the cells, treat, and then visualize? Are they then studying the fate of old PG? How does BADA get incorporated into PG in V.cholerae? Is it through LDT activity or some other way? Without more explanation, it is hard to interpret the results.
BADA does get incorporated through either LDT or PG synthesis activity in V. cholerae, but for these experiments, the specific incorporation pathway is inconsequential, since we only focus on the end product (stained PG). We think that what we visualize is not the fate of old PG (otherwise we would see similar strong stains with Fosfomycin, which inhibits cell wall synthesis upstream of PG strand generation by PBPs/SEDS), but rather visualizes the generation of long, uncrosslinked PG strands due to the inhibition of PBP transpeptidase activity. We have added more explanations of this assay to the revised manuscript.
The last conclusion is not supported by data. There are no data showing that LTG activity is temporally separate from PG synthesis.
We would like to point out that this is not framed as a conclusion per se, but rather a plausible speculation. Our data showing soluble strand accumulation in the WT strongly suggest that LTGs do not work in perfect harmony with synthesis, but rather degrade strands AFTER they accumulate (i.e., temporally separate). We further believe that complementation with a heterologous enzyme (MltE), which does not have a homolog in V. cholerae strongly argues that LTGs and PG synthesis do not have to associate through protein-protein interactions. All this adds to an emerging model that PG synthesis and LTG-mediated degradation are not as tightly co-ordinated as one might assume.
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Evaluation Summary:
This study addresses a major missing element in the understanding of how bacteria grow their cell wall and the role of lytic transglycosylases in this process. It had been previously assumed these enzymes cut glycan strands to make room for the insertion of new glycans. However, results presented in this manuscript demonstrate these enzymes have a very different, yet essential role in degrading uncrosslinked glycan strands in the periplasm. The authors further demonstrate that in the absence of lytic transglycosylases, cells undergo periplasmic stress due a toxic accumulation of these "free strands" in the periplasm. The work will be of interest to those in the bacterial growth and division field.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also …
Evaluation Summary:
This study addresses a major missing element in the understanding of how bacteria grow their cell wall and the role of lytic transglycosylases in this process. It had been previously assumed these enzymes cut glycan strands to make room for the insertion of new glycans. However, results presented in this manuscript demonstrate these enzymes have a very different, yet essential role in degrading uncrosslinked glycan strands in the periplasm. The authors further demonstrate that in the absence of lytic transglycosylases, cells undergo periplasmic stress due a toxic accumulation of these "free strands" in the periplasm. The work will be of interest to those in the bacterial growth and division field.
(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. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors used a combination of multiple gene knock-outs of lytic transglycosylsases with extensive phenotyping of the mutant strains to conclude that the essential role of Ltgs for bacteria is to reduce periplasmic crowding with soluble macromolecular fragments of peptidoglycan. The extensive phenotyping is based on complementation assays, growth phenotypes, morphological phenotypes, analysis of suppressor phenotypes, sensitivity to accumulation of periplasmic polysaccharides and to antibiotics promoting the peptidoglycan futile cycle, and finally, analysis of soluble peptidoglycan accumulated in the periplasm.
The work is well written, easy to follow and technically sound. Collectively, it provides circumstantial evidence for the authors final model.
This work supports previous seminal work on the futile …
Reviewer #1 (Public Review):
The authors used a combination of multiple gene knock-outs of lytic transglycosylsases with extensive phenotyping of the mutant strains to conclude that the essential role of Ltgs for bacteria is to reduce periplasmic crowding with soluble macromolecular fragments of peptidoglycan. The extensive phenotyping is based on complementation assays, growth phenotypes, morphological phenotypes, analysis of suppressor phenotypes, sensitivity to accumulation of periplasmic polysaccharides and to antibiotics promoting the peptidoglycan futile cycle, and finally, analysis of soluble peptidoglycan accumulated in the periplasm.
The work is well written, easy to follow and technically sound. Collectively, it provides circumstantial evidence for the authors final model.
This work supports previous seminal work on the futile cycle of peptidoglycan synthesis in the presence of beta-lactams but by extending it to homeostasis and not only during cell envelop stress.
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Reviewer #2 (Public Review):
The paper "Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products" by Weaver et al. reveals multiple findings, some of which are transformative for the cell wall field. 1) First and foremost, they finally nail down the role of lytic transglycosylases in the evolution of the cell wall, revealing that they cleave and process uncrosslinked glycans arising from endopeptidase activity. 2) They also document the periplasmic stress that occurs when these strands are not degraded, raising interesting future questions about the role of glycans in the resistance to osmotic stress and perhaps the balance of turgor pressure in the cytoplasm. 3) This work also suggests that the lytic transglycosylases are separate from the synthetic enzymes, further adding to the …
Reviewer #2 (Public Review):
The paper "Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products" by Weaver et al. reveals multiple findings, some of which are transformative for the cell wall field. 1) First and foremost, they finally nail down the role of lytic transglycosylases in the evolution of the cell wall, revealing that they cleave and process uncrosslinked glycans arising from endopeptidase activity. 2) They also document the periplasmic stress that occurs when these strands are not degraded, raising interesting future questions about the role of glycans in the resistance to osmotic stress and perhaps the balance of turgor pressure in the cytoplasm. 3) This work also suggests that the lytic transglycosylases are separate from the synthetic enzymes, further adding to the "disconnected" model of the enzymes that synthesize and process the cell wall.
I very much appease a large amount of work done here and that the authors were able to infer some sort of "cumulative damage" from their initial experiments, and then figure out the non-obvious cause behind these phenomena.
The experiments are all well documented, with a large amount of data supporting their claims, all of which appear well supported, and the more speculative claims are phrased as such.
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Reviewer #3 (Public Review):
In this manuscript the authors make several conclusions, according to the abstract:
1 - LTG activity is essential by contributing to a process independent of PG recycling.
2 - LTGs are important because of their catalytic activity rather than because of a protein-protein interaction.
3 - LTG mutants are hypersusceptible to production of periplasmic polymers.
4 - LTGs prevent toxic periplasmic crowding and their function is temporally separate from PG synthesis.The authors perform a series of genetic experiments that lead to their conclusions. Their first conclusion is well supported by data showing that a PG recycling mutant does not have the same defects as their LTG mutant.
Their second conclusion needs more justification/explanation. They show a catalytic mutant of RlpA is unable to sustain growth as the …
Reviewer #3 (Public Review):
In this manuscript the authors make several conclusions, according to the abstract:
1 - LTG activity is essential by contributing to a process independent of PG recycling.
2 - LTGs are important because of their catalytic activity rather than because of a protein-protein interaction.
3 - LTG mutants are hypersusceptible to production of periplasmic polymers.
4 - LTGs prevent toxic periplasmic crowding and their function is temporally separate from PG synthesis.The authors perform a series of genetic experiments that lead to their conclusions. Their first conclusion is well supported by data showing that a PG recycling mutant does not have the same defects as their LTG mutant.
Their second conclusion needs more justification/explanation. They show a catalytic mutant of RlpA is unable to sustain growth as the only LTG in the cell. However, I am confused by their wording around RlpA in general. In the text they note that their delta_7 mutant, which encodes RlpA, 'has no highly active LTGs' (lines 130-131). Does that imply that RlpA is not an LTG? In the discussion they note that E.coli RlpA has no LTG activity. Is this enzyme known to have LTG activity in V.cholerae? One important control would be to show that the catalytically inactive protein is stable (i.e. that the defect is not due to protein misfolding). This could be supported by looking at protein stability via Western or even quantifying the fluorescence data in Figure S3b.
Their third conclusion also needs more support. The authors do a series of experiments showing that delta7 is more susceptible to SacB. What are the data that show sacB produces large polysaccharides molecules in the periplasm rather than (or in addition to) the cytoplasm? This would be important to show as these data are the main test of the authors model.
The authors have other data that all argue for their model that LTG deficient strains have an excess of periplasmic crowding. The suppressor of delta_opgH is intriguing, but does not restore the morphological defects in delta_7, suggesting that the increase in length during prolonged growth may not be caused by periplasmic crowding, or at least is not alleviated by deletion of OpgH. What then does the deletion of OpgH suppress? Here, I was confused by the experiments in low salt. The authors write that the cells lyse (line 222) but this is not shown anywhere. Growing the cells continually in low salt may not be the hypoosmotic challenge the authors presume. A challenge typically implies an acute change in osmolarity, rather than a prolonged exposure, which may allow cells to adapt.
I was also highly confused by the antibiotic + BADA staining experiments. Do the authors stain the cells, treat, and then visualize? Are they then studying the fate of old PG? How does BADA get incorporated into PG in V.cholerae? Is it through LDT activity or some other way? Without more explanation, it is hard to interpret the results.
The last conclusion is not supported by data. There are no data showing that LTG activity is temporally separate from PG synthesis.
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