Lipopolysaccharide integrity primes bacterial sensitivity to a cell wall-degrading intermicrobial toxin

  1. Kristine L Trotta
  2. Beth M Hayes
  3. Johannes P Schneider
  4. Jing Wang
  5. Horia Todor
  6. Patrick Rockefeller Grimes
  7. Ziyi Zhao
  8. William L Hatleberg
  9. Melanie R Silvis
  10. Rachel Kim
  11. Byoung Mo Koo
  12. Marek Basler
  13. Seemay Chou

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Abstract

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the molecular basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa , Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, surface lipopolysaccharide, that modulate Tae1 toxicity in vivo . Disruption of lipopolysaccharide synthesis provided Escherichia coli (Eco) with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study highlights the consequences of co-regulating essential pathways on recipient fitness during interbacterial competition, and how antibacterial toxins leverage cellular vulnerabilities that are both direct and indirect to their specific targets in vivo .

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  1. Version published to 10.1101/2023.01.20.524922v3 on bioRxiv
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    Reply to the reviewers

    We thank the reviewers for their constructive feedback on our manuscript. They did a very comprehensive and helpful job of laying out some key areas that could be improved. We were heartened by the fact that there was a fair amount of overlap between the two reviewers, and that comments were largely addressable without further experimentation.

    Below, we provide a summary of how we have attempted to address the comments and concerns from both reviewers. We also provide the rationale and action items for our responses. Overlapping comments from both reviewers have been consolidated and responded to together.

    Comment 1 (Reviewer #1, Minor Comment 1 & Reviewer #2, Significance)

    Both reviewers raised concerns about our choice to focus on essential genes in our CRISPRi screen, which could potentially underestimate the role of non-essential factors contributing to Tae1 sensitivity or resistance.

    Rationale: We agree with the reviewers that including non-essential genes could provide additional insights into the roles of non-essential factors in Tae1 sensitivity and resistance. We believe our focus on essential genes contributes a unique perspective to the field, as there already exists a body of work that interrogates non-essential genes in this space. Here are some citations that represent this body. We will highlight these better in the manuscript.

    Lin, H.-H.; Yu, M.; Sriramoju, M. K.; Hsu, S.-T. D.; Liu, C.-T.; Lai, E.-M. A High-Throughput Interbacterial Competition Screen Identifies ClpAP in Enhancing Recipient Susceptibility to Type VI Secretion System-Mediated Attack by Agrobacterium Tumefaciens. Front Microbiol 2020, 10, 3077. https://doi.org/10.3389/fmicb.2019.03077.

    Hersch, S. J.; Sejuty, R. T.; Manera, K.; Dong, T. G. High Throughput Identification of Genes Conferring Resistance or Sensitivity to Toxic Effectors Delivered by the Type VI Secretion System; preprint; Microbiology, 2021. https://doi.org/10.1101/2021.10.06.463450.

    Additionally, our screen was experimentally optimized for essential genes using our approach. The knockdown strategy is useful specifically for essential genes because E.coli is phenotypically very sensitive to essential gene perturbations (see more here: https://doi.org/10.1128/mBio.02561-21). While it would have been ideal to include non-essential genes too, doing so would require a different additional optimization that we believe would have diluted our bandwidth for this study. We do thank the reviewers for recognizing how much effort went into this!

    We do acknowledge this is a limitation and want to make sure the readership is aware of that. Ideally, one could do more rigorous side-by-side comparisons between studies if the approaches, set-up, and assays are the same. Unfortunately, due to differences in experimental set-up, we could not directly compare with the non-essential screens. We hope others will pick up where we left off. Here are some action items we can take to increase the odds of that:

    In the Introduction, we will mention other studies and highlight the need to investigate essential genes side-by-side with non-essential. (Lines 64-7) In the Discussion, we will add a sentence that acknowledges the importance of exploring non-essential genes for a more comprehensive understanding of Tae1 sensitivity and resistance. (Lines 484-5)

    Comment 2 (Reviewer #1, Minor Comment 5 & Reviewer #2, Major Comment)

    Both reviewers mentioned that the dormancy state in msbA-KD cells is not well characterized and its relationship with Tae1 resistance is not convincingly shown.

    Rationale: We agree that our manuscript does not clearly pin down whether Tae1 resistance is linked to a true dormancy state. There are some intriguing similarities between what we observe and what is classically known as “dormancy” or “persistence”, which have specific definitions. Although we don’t yet have a concrete reason to think it’s NOT those states, we also don’t have sufficient data to point to it clearly being the same at a mechanistic or cellular level. This is merely a hypothesis that our work suggests. We would love to see others follow up on this, as we suspect there are overlaps and potentially additional cellular states that have yet to be clearly defined in this field of bacterial physiology.

    Here is how we propose to address this concern:

    We simplified our language to be more descriptive and less loaded in terms of nomenclature around dormancy or persistence. Namely, we are referring to the cells in a more descriptive way with “slowed growth.” This allows us to clearly describe what we observe without attempting to ascribe mechanism or anything beyond that. It doesn’t fundamentally change the overarching interpretation of our study. (Lines 444, 490,497-9) In the Discussion, we will add text emphasizing the need for follow-up studies to fully address whether there is indeed a connection between Tae1 resistance and slowed growth. (Lines 491-3)

    Comment 3 (Reviewer #2, Major Comment)

    The reviewer asks if the degradation of the sugar backbone is also required for lysis or if it is just the crosslinking step that is important.

    Rationale: This is an astute point. We acknowledge that the degradation of the sugar backbone may play a role in lysis, and it’s predicted that this may be why the Pae H1-T6SS delivers a second PG-degrading toxin (Tge1), a muramidase that targets the sugar backbone. The most parsimonious conclusion from past studies by us and others is that Tae1 is critical for lysis, but not sufficient in the absence of any backbone-targeting enzyme. Indeed, many T6SS-encoding bacterial species also encode >1 type of PG-degrading enzyme, which may speak precisely to the reviewer’s point. However, it should also be noted that there may be endogenous enzymes with activities that can be leveraged alongside these toxins for the same effect.

    Action items:

    In the Discussion, we will add a sentence addressing the potential role of sugar backbone degradation in the lysis process and the need for future research on this topic. (Lines 524-6)

    Comment 4 (Reviewer #1, Minor Comment 2)

    The reviewer asks why lptC-KD leads to sensitivity to Tae1, while msbA-KD leads to resistance, considering both genes are implicated in LPS export.

    Rationale: We appreciate the reviewer's careful attention to the underlying biology. They are absolutely correct in pointing this difference out. Our interpretation is that the different phenotypes may indicate that although the LPS biosynthesis superpathway intersects with PG synthesis, lptC and msbA may intersect with PG synthesis in distinct ways. We can address this concern through the following:

    We will add a sentence in the Discussion section providing our interpretation of the different phenotypes observed for* lptC-KD* and msbA-KD. (Lines 508-13)

    Comment 5 (Reviewer #1, Minor Comment 4)

    The reviewer notes that the contribution of* msbA* to Tae1 resistance appears minor based on the results in Figure 3d.

    Rationale: There are actually two aspects to this concern, which we note below. We found it difficult to fully capture it in the manuscript, but our thoughts are as follows.

    (1) Technical viewpoint:

    Bacterial competition experiments are inherently noisy. The quantitative read-out is easily impacted by a number of parameters, including cellular density, input ratio between competitor cell types, growth stage, and possibly other environmental factors that are difficult to predict. In general, our view is that we should avoid over-indexing on the degree of the phenotype, focusing more on the direction of the phenotype (loss of statistically-significant Tae1 sensitivity) and the fact that it is reproducible in our hands. Furthermore, our argument is bolstered by clear validation of the loss of Tae1 sensitivity through orthogonal lysis assays (Fig. 4a-c).

    (2) Biological viewpoint

    It is challenging to isolate the specific interaction between Tae1 and individual genetic determinants, as we think it’s a complex system with multiple factors simultaneously at play. It is crucial to acknowledge that the unique contribution of Tae1 is only a part of the T6SS. There may be other compensatory actions that influence the outcomes observed, such as upregulation of non-Tae1 toxins, regulation of system activation/firing, timing and location of T6S injections, etc. We think these are exciting possibilities and that more groups should delve into the context-dependent dynamics of the system. Although outside the scope of our manuscript, we would be open to suggestions for how we can further emphasize this point.

    Comment 6 (Reviewer #2, Minor Comment)

    The reviewer recommends that we discuss whether our findings are specific to Tae1 or if they can be extrapolated to other toxins.

    Rationale: We understand the reviewer's interest in understanding the broader implications of our findings. Although our study focuses specifically on Tae1, we believe that our findings may provide insights into the mechanisms of sensitivity and resistance to other toxins that target the cell wall. However, experimentally investigating this would fall outside the scope of our current manuscript.

    Additional Minor Revisions

    Table 1: I would label MsbA and LptC as "LPS transport" and not "LPS synthesis" (Reviewer 1) Rationale: We agree that using “LPS transport” to describe the gene functions for lptC and msbA is more specific to their functions.

    Table 1 was updated to change the “pathway/process” categorizations for lptC and msbA from “LPS synthesis” to “LPS transport”. In line with this comment, we also changed the pathway/process categorization for murJ (Lipid II flippase) to “PG transport”. Figure 3 legend: "...deformed membranes .........are demarcated in (g) and (h)" (Reviewer 1) We thank the reviewer for pointing out the missing text in this figure legend.

    We corrected the error by adding the missing text back in Figure 3. Line 339-341: Supp. Fig. 9 should be Supp. Fig. 8 (Reviewer 1) *Referenced Supp. Fig. was corrected. * Second, (L422-425) the authors conclude that their data demonstrate a "reactive crosstalk between LPS and PG synthesis". I disagree. There is no information in the paper that this is the case. The authors can only suggest that cross talk may occur. (Reviewer 2) *We agree. Line 421-2: replaced “demonstrate” with “suggest” to soften the argument. *

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    This study reports the finding that lipopolysaccharide integrity modulates bacterial sensitivity to a Type-6-secreted bacterial toxin. The authors used the Tae1 amidase produced by the P. aeruginosa T6SS and Escherichia coli bacteria as prey cells as a model system to test the effect of knockdowns in essential gene expression of the prey. This was accomplished by constructing a library of knockdown (KD) genes based on Crispr/Cas9 and selecting for those targets where E. coli prey is not killed. The screen revealed, as expected, that KD genes encoding cell wall synthesis assembly (and bamA, involved in OM protein assembly) enhanced the sensitivity to Tae1. In contrast, KD targets in genes involved in lipid metabolism and lipopolysaccharide synthesis conferred resistant to the amidase toxin. The authors hypothesized that non-PG components of the cell envelope may shape Tae1 toxicity and undertook a more detailed analysis of the effects of knocking down one of these genes, msbA, using a various biochemical and imaging approaches. The MsbA protein is an ATPase permease that plays an essential role in flipping newly synthesized lipid A across the bacterial inner membrane. The authors show that resistance to Tae1 in msbA-KD is independent of cell wall hydrolysis (meaning that the Tae1 remains active), PG synthesis is suppressed (despite PG is still Tae1 sensitive), and that protein synthesis and growth is suppressed. This latter observation suggests that the E. coli prey enters a persistent (dormant) state that protects it from Tae1 toxicity. The authors conclude that Tae1 susceptibility in vivo is determined by cross talk between essential cell envelope pathways and the general growth state of the cell.

    Major comments:

    This is a nice study unravelling cellular off target factors that affect the killing in vivo by a T6SS toxin. In that sense the study is novel since the interplay of T6SS effectors in the context of the physiological state of the prey cell has not been directly investigated. so this study adds new information to the literature in the field.

    I have several comments concerning the interpretation of the results.

    First, it is interesting that Tae1, being an amidase, can be the sole responsible for PG degradation. The enzyme cleaved the peptide bridges but has no effect on the PG backbone. The study was not designed to pick up autolysins (since only essential genes were targeted) but one would assume that degradation of the sugar backbone must also be required for lysis.

    Second, (L422-425) the authors conclude that their data demonstrate a "reactive crosstalk between LPS and PG synthesis". I disagree. There is no information in the paper that this is the case. The authors can only suggest that cross talk may occur.

    Third, Tae1 maximal effect is present when new PG is made, which also begs the question about the location of this protein in the PG mesh. Like B-lactam and other PG-active antibiotics, the effect of Tae1 requires active cell growth. This is also consistent with the authors' finding that the msbA-KD bacterial cells enter a state of dormancy or persistence, which will make them capable of overcoming Tae1 toxicity.

    Fourth, an important outcome of protein synthesis inhibition and PG synthesis is increased oxidation and lipid peroxidation. This could also influence the results obtained in this study. It would be consistent with the other targets observed, which compromise lipid metabolism and membrane trafficking and secretion.

    Referees Cross-commenting

    Based on my own review and that of Reviewer 1, I think we both agree that there are 2 major limitations in this work: (i) the KD library only targets essential genes and this would potentially miss non-essential genes that when targeted for mutated could lead to synthetic lethal phenotypes that could be more revaling than a general defect protein synthesis, etc. and (ii) the dormancy state is not well characterized.

    Despite these points the study is very nicely done with a huge amount of work.

    Significance

    This is an important study addressing experimentally the complexities of bacteria-bacteria interactions in the context of predator-prey interplay. The T6SS effectors affecting PG appear to have the same characteristics as known antibiotics and bacteria use similar strategies to protect themselves from PG attack. This is not only to increase growth as an escape approach but also to reduce it to a point in which the target cell cannot be effectively killed despite the presence of the toxin.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The manuscript by Trotta and co-workers investigates functions that shape E. coli envelope when cells are targeted by the cell-wall degrading toxin Tae1. The experimental setting employed by the authors is well thought and is based on the competition engaged by P. aeruginosa (Pae) expressing the type 6 secretion system (T6SS) against E. coli cells. In this context, the authors used an arrayed library of chromosomally encoded CRISPRi strains targeting essential genes of E. coli (knockdowns, KDs) to identify functions that increase or decrease E coli fitness following interbacterial competition with Pae cells expressing Tae1. The majority of genes whose depletion makes E. coli cells more sensitive to the toxin are implicated in PG synthesis while depletion of genes implicated in other cell envelope processes can result in toxin sensitivity or resistance. Among genes whose depletion makes E. coli cells more resistant to the toxin the authors selected those implicated in LPS biogenesis (msbA-KD and lpxK-KD), to investigate the hypothesis that non-PG components of the cell envelope may also shape Tae1 toxicity. While resistance to lpxK-KD to Tae1 could not be validated in the reconstructed strain likely due to a polar effect, the reconstituted msbA-KD gained Tae1 specific resistance. Further characterization of the msbA-KD revealed that Tae1 resistance is independent of cell wall hydrolysis and PG dynamics. By showing that both slow growth and decreased protein synthesis is specifically linked to Tae1 resistance in msbA-KD cells the authors suggest that a persistent state, induced by block of LPS biogenesis, helps depleted msbA cells to resist the toxic activity of Tae1. Overall, the experimental approach is solid, the developed in vivo screen to identify genetic interaction between secreted Tae1 and E coli is smart and well thought. I also acknowledge the huge work performed by the authors to characterize selected KD strains.

    My few comments to the manuscript are reported below.

    Major Comments

    There are no major comments

    Minor Comments

    1. Why the authors limit the search of functions that help P. aeruginosa to antagonize E. coli cells only to essential genes? I understand that the available CRISPRi strains collection (developed by Carol Goss and co-workers) is only targeting essential genes, but the rationale for this choice should be discussed. This approach is inevitably underestimating the role of perhaps important non-essential factors contributing to Tae1 sensitivity/resistance.
    2. It is intriguing that in Table 1 lptC is listed among the genes whose depletion leads to sensitivity to Tae1 whereas msbA transcriptional down regulation leads to resistance. Both LptC and MsbA are implicated in LPS export to the cell surface, and one would expect that their down-regulation leads to similar output in competition experiments with P. aeruginosa. Can the authors comment on that?
    3. Based on results reported in Figure 3 d (fold change msbA-KD CFU) the contribution of MsbA to Tae1 resistance seems minor. Can the authors comment?
    4. Based on the observation that msbA-KD cells arrest growth, do not divide, and decrease protein synthesis, the authors suggest that these cells enter a persistent state which could protect against Tae1 activity by passive tolerance. In support of this hypothesis the authors refer to published work (Roghanian et al. PloSOne 2019) showing that CRISPRi KD in lpxA (the first gene of the LPS biosynthetic pathway) triggers a dormancy state to respond to imbalances in outer membrane biogenesis. In this manuscript Roghanian and co-workers show that CRISPRi KD in lptA (encoding the periplasmic component of the LPS export machinery) share the same phenotypes as lpxA. These observations bring me again to the comment n. 2 above. I think that the authors should comment on this. In my opinion this is the weakest part of the manuscript as it is not convincingly showed i) that msbA-Kd cells enter a dormancy state, ii) how this dormancy state is related to Tae1 resistance.

    Table 1: I would label MsbA and LptC as "LPS transport" and not "LPS synthesis"

    Minor points

    Figure 3 legend: "...deformed membranes .........are demarcated in (g) and (h)"

    Line 339 341: Supp. Fig. 9 should be Supp. Fig. 8

    Significance

    This study aims at understanding how Tae1, a PG-degrading toxin secreted by T6SS specifically aids P. aeruginosa in antagonizing E. coli cells in vivo. By exploiting a smart in vivo genetic screen, the authors want to understand at the molecular level the interplay between Tae1 and essential functions in E. coli. The study of interspecies competition offers the possibility to investigate and dissect complex physiological processes and the interactions between them. The work is solid and the experimental plan well-conceived. However, the in vivo genetic screen is limited to the search of essential functions implicated to sensitivity or resistance to the secreted toxin. Such an approach is inevitably underestimating the role of perhaps important non-essential factors contributing to Tae1 sensitivity/resistance. Also, as indicated above, I think that the authors did not convincingly show i) that msbA-Kd cells enter a dormancy state, ii) how this dormancy state is related to Tae1 resistance.

    Audience Broad audience / basic research

    Expertise in outer membrane biogenesis

  5. Version published to 10.1101/2023.01.20.524922v2 on bioRxiv
  6. Version published to 10.1101/2023.01.20.524922v1 on bioRxiv