Antibacterial potency of type VI amidase effector toxins is dependent on substrate topology and cellular context

  1. Atanas Radkov
  2. Anne L Sapiro
  3. Sebastian Flores
  4. Corey Henderson
  5. Hayden Saunders
  6. Rachel Kim
  7. Steven Massa
  8. Samuel Thompson
  9. Chase Mateusiak
  10. Jacob Biboy
  11. Ziyi Zhao
  12. Lea M Starita
  13. William L Hatleberg
  14. Waldemar Vollmer
  15. Alistair B Russell
  16. Jean-Pierre Simorre
  17. Spencer Anthony-Cahill
  18. Peter Brzovic
  19. Beth Hayes
  20. Seemay Chou

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Abstract

Members of the bacterial T 6SS a midase e ffector (Tae) superfamily of toxins are delivered between competing bacteria to degrade cell wall peptidoglycan. Although Taes share a common substrate, they exhibit distinct antimicrobial potency across different competitor species. To investigate the molecular basis governing these differences, we quantitatively defined the functional determinants of Tae1 from Pseudomonas aeruginosa PAO1 using a combination of nuclear magnetic resonance and a high-throughput in vivo genetic approach called deep mutational scanning (DMS). As expected, combined analyses confirmed the role of critical residues near the Tae1 catalytic center. Unexpectedly, DMS revealed substantial contributions to enzymatic activity from a much larger, ring-like functional hot spot extending around the entire circumference of the enzyme. Comparative DMS across distinct growth conditions highlighted how functional contribution of different surfaces is highly context-dependent, varying alongside composition of targeted cell walls. These observations suggest that Tae1 engages with the intact cell wall network through a more distributed three-dimensional interaction interface than previously appreciated, providing an explanation for observed differences in antimicrobial potency across divergent Gram-negative competitors. Further binding studies of several Tae1 variants with their cognate immunity protein demonstrate that requirements to maintain protection from Tae activity may be a significant constraint on the mutational landscape of tae1 toxicity in the wild. In total, our work reveals that Tae diversification has likely been shaped by multiple independent pressures to maintain interactions with binding partners that vary across bacterial species and conditions.

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  1. Version published to 10.7554/elife.79796 on eLife
  2. Review Commons

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    Reply to the reviewers

    Two reviewers commented on the smeared appearance of Tae1 bands in our Western blot analyses (Figure 4F and 5B) and asked us to improve their technical quality.

    -We agree and will repeat these experiments with more careful attention to lysate preparation, using a higher percentage SDS gel for better separation of low molecular weight proteins as suggested.

    Reviewer 2 requested that we assess how Tae1 variants impact interbacterial competition outcomes.

    -We agree that this would be interesting to take a look at. While this will not be feasible for every variant we examine in the paper, we can conduct comparative interbacterial assays between *P. aeruginosa *and E. coli using P. aeruginosa strains with a *tae1 *point mutation for c110s. Given that our biochemical experiments show that this hyperactive variant evades inhibition by the cognate immunity protein, we expect that this may decrease *P. aeruginosa *fitness, even in the context of competition.

    More generally, we think that examining Tae1 variants in the context of interbacterial competitions would be a critical orthogonal approach in order to validate that the DMS results have any bearing on competition outcomes. However, we feel that major focus of this paper is on the more molecular and biophysical insights that our approach can offer. Our study tests our assumptions about the kinds of features and surfaces that are important for proteins that engage with non-canonical complex substrates. It is, of course, interesting to think about the implications of this for physiological phenotypes and the drivers of toxin evolution. It is also exciting to imagine how this kind of information could be used to one day engineer certain interbacterial outcomes. We hope that others in the field will push our efforts into these directions, but we do not feel that these directions are essential for our conclusions. However, our conclusions on the molecular and biophysical aspects have helped generate interesting hypotheses in microbial ecology that could be largely followed up on by others.

    In order to conduct well-controlled P. aeruginosa:E. coli competition assays for more Tae1 variants, we would need to generate a significant number of new P. aeruginosa strains encoding point mutations for each of our variants across several genetic backgrounds. The competitions themselves also require a considerable amount of work to optimize and quantify. We are able to do this for one of the variants as previously mentioned (C110S). It’s important to note that the first author of this paper, who was the primary driver of this work, is no longer in my lab or in academia. As for myself, I am also in the middle of a transition out of academia and am actively ramping down my lab at UCSF. I no longer have the space or appropriate set-up to support this longer-term effort.

    Reviewer 2 asked that we examine Tae1 (WT and C110S) expression levels in vivo to more precisely examine whether increased self-intoxication by Tae1C110S in P. aeruginosa was due to differences in toxin activity or toxin levels.

    We agree with this suggestion and will look at toxin protein levels by Western blot analysis in the context of P. aeruginosa cells grown 1) alone on solid media and 2) together with E. coli on solid media during interbacterial competition using conditions that match our other competition assays.

    All 3 reviewers asked us to provide more experimental evidence addressing the hypothesis that differential peptidoglycan (PG) affinity across Tae1 variants could explain variation in toxic activity.

    -We agree that this is an interesting point to follow up on further. To be clear, we also do not know whether this hypothesis is true at this stage, and the answer is not necessarily critical for our central advance, but we would like to give it a try! We have devised an approach to ask the question experimentally across a subset of our deep mutational scanning (DMS) variants.

    Reviewer 1 suggested that we quantify *in vitro *binding affinities for PG using isothermal titration calorimetry (ITC). However, given that ITC requires high concentrations of well-defined homogeneous substrates, which we are not able to generate for more complex higher order structures of cell wall PG, we propose a pull-down based approach.

    Briefly, we plan to conduct pull-downs using insoluble, purified cell wall sacculi from our two E. coli grown under the two conditions as bait for recombinant Tae1 proteins. Given that intact sacculi or inherently insoluble, we can simply collect bound Tae1 through centrifugation of sacculi pellets and examine the amount of Tae1 associated by Western blot analysis. These analyses will need to be conducted across a titration of Tae1 concentrations and also with catalytic activity inhibited to avoid solubilization of sacculi. We will block Tae1 hydrolysis by carrying out pull-downs in the presence of a general commercially-available cysteine hydrolase inhibitor, E64. If there is indeed differential affinity for PG underlying lytic differences across Tae1 variants, we would expect to see greater relative association of Tae1 variants with the type of cell wall sacculi that they more effectively lyse in our DMS screen. We would expect the reverse trend to also be true (lower affinity for less active variants).

    Reviewer 1 would like to know if we have done lysis experiments with any E. coli mutants that only impact PG density but not PG polymer structure? If they haven’t tested any E. coli mutants, have we done lysis experiments using drugs that have a similar impact on PG? Even if we don’t include these data in the paper, the reviewer would like us to comment on the trends we have observed.

    We have not done experiments in any mutants or chemical backgrounds known to only impact PG density but not polymer structure. We think this would be a very interesting angle! But unfortunately this is outside the scope of this study. It would require that we first experimentally confirm that the restrictive effect on only density is clearly demonstrated using a variety of techniques, including microscopy, chemical analyses, and biophysical probing of sacculi.

    Reviewer 1 asked for additional DMS screens in more conditions

    We love this idea! In fact, we hope that others are motivated to adopt our workflow to run many more DMS screens for T6S toxins, as we believe these screens provide a lot of useful and sometimes surprising insights that could be of great interest to others. However, we believe that the primary goal of this paper is to establish this methodology as a compelling approach for studying toxins and, more generally, proteins with complex cellular substrates. It does not necessarily fall within the scope of this paper to fully assess the mechanistic implications of cell wall diversity across a wide range of conditions.

    In our experience, rigorously conducting DMS screens requires a significant amount of effort and resources to establish consistent experimental conditions. Also, a non-trivial number of costly sequencing-based experiments are required across control and variables for the results to be statistically sound and meaningful. Furthermore, experimental validation of results are ultimately important for our ability to confidently generate hypotheses stemming from these datasets. As stated above, the first author of this paper, who was the primary driver of this work, is no longer in my lab or in academia. As for myself, I am in the middle of a transition out of academia and am actively ramping down my lab at UCSF. I no longer have the space or appropriate set-up to support this longer-term effort.

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    Referee #3

    Evidence, reproducibility and clarity

    This paper by Radkov et al. represents an extensive structure-function-evolution treatment of the Type VI secretion effector protein Tae1. Using mutational scanning, the authors identify multiple residues that either enhance or reduce Tae1 function in an E. coli model, and validate these residues through direct functional assays. The main conclusion is that Tae1 contains a surprising number of non-intuitive residues important for its activity, particularly several surface-exposed residues far from the active site. The authors then suggest that these residues mediate binding to specific PG architectures and supply some evidence that the functional mutation landscape changes when the DMS assays is repeated in E. coli with altered cell wall architecture. Lastly, natural variants of Tsae1 are identified and discussed in the context of the trade-off between optimal toxicity and maintenance of self-immunity.

    I have no major comments. The study is beautifully-done, with all controls in place. It might be worth following up on their putative PG binding residue mutants with an additional binding assay (MST, or just a crude cell wall pulldown assay), but that is not critical to support the main conclusions.

    Minor comments

    • The Western Blot of the vector control in Fig. 4F has the same impurities as the one in Fig. 5 B. Was the control blot re-used? If so, please indicate in the figure legend. Also, please show full Western Blots in supplemental material.
    • Small typo in Fig. 5 legend ("does is not")
    • The citation in line 108 seems a little off - that does not seem to support a physiologically relevant context for Tae.
    • Line 122/123 - something seems to be missing in this sentence.
    • Line 148 - this is not clear to me. Did they sequence plasmid barcodes (are those in the plasmid backbone?), or the mutated orfs?

    Significance

    This paper makes an impactful contribution to the open question of substrate- and species-specificity of PG hydrolases, particularly those weaponized by Type VI secretion systems. The major advance here is that PG binding by the hydrolase, and PG architecture of the substrate, are important determinants of Tae function and that this has important evolutionary consequences. The study will be of interest to the Type VI secretion community, but also to the PG turnover field.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    This manuscript aims to investigate the molecular basis underlying the differential toxicity of the bacterial T6SS amidase effector (Tae) by using Pseudomonas aeruginosa Tae1 as a model. The rationale is that while Tae is a conserved T6SS toxin degrading bacterial peptidoglycan (PG) by specific cleavage activity, different Tae toxins of the same family exhibit distinct lysis/antibacterial activity. Thus, the authors used combination of a deep mutagenesis scanning (DMS) coupled with fitness assay, NMR, and PG-binding/amidase activity to address this question by expressing Tae1 variants in E. coli. Besides finding the residues at/near the catalytic site critical for amidase activity, the authors further discovered many surface-exposed resides distant from the catalytic cleft also contributed to Tae activity likely by affecting binding or hydrolysis of PG. The authors further explored whether the residues contributing to loss or gain of Tae1 activity could be different against different PG structure by performing the same suite of DMS analyses from E. coli grown in the presence of D-Met, which resulted in reduced PG density and crosslinks. They discovered the fitness landscape of Tae1 variants shift dramatically, suggesting that Tae1 toxicity is highly context-dependent and optimizable for specific PG forms. A hyperactive Tae1 C110S variant is also naturally encoded in a subset of Proteobacteria outside of P. aeruginosa. This further led to a prediction that Tae1 C110S variant may evade binding and inhibition by cognate immunity, which was confirmed by the higher binding affinity of WT Tae1 than C110S Tae1 determined by ITC analysis. Together, the authors concluded that substrate-specificity and toxin-immunity interactions are the two distinct selective pressures for shaping diversity across the Tae1 toxin superfamily.

    Major comments:

    This is a well thought, carefully designed and executed research article reporting important and interesting findings. The conclusions made are mostly supported by the provided data. However, the toxicity assay for Tae1 variants except C110S was only validated by ectopic expression in E. coli or in vitro activity assay. Considering Tae1 is a bacterial toxin involved in interbacterial antagonism, the mutants with newly discovered key residues contributing to loss or gain of function shall be also evaluated for their role in the context of interbacterial competition, not simply by the cell lysis assay of expressed Tae1 variants in E. coli. Below are the specific comments that shall be addressed in order to claim the findings of this work .

    1. It is an exciting finding that several surface residues distal from catalytic core mediate PG hydrolysis or binding. While the validation of their cell lysis activity by expressing each Tae1 variants fused with LepB signal peptide is informative, the role of these surface residues in toxicity shall be also tested by interbacterial competition assay either using E. coli or susceptible Pseudomonas aeruginosa strain as a prey. Tai may be expressed in E. coli prey to determine its neutralization activity during interbacterial competition context.
    2. Based on the results that fitness landscape of Tae1 variants grown in the presence or absence of D-Met, the authors stated in line 334 "Condition-specific phenotypes suggests that Tae1 toxicity in vivo is highly context-dependent and optimizable for specific PG forms." However, there could be other physiological changes due to D-methionine. To claim this, the authors may test the surface residues with altered impacts on fitness between two growth conditions for their PG-binding activity using PG isolated from culture in the presence or absence of D-Met .
    3. Quality of western blotting for Tae1 variants in Fig. 4F, 5B should be improved as the signals from WT is not clearly detected for comparison. The authors may use higher percentage of SDS-PAGE for better resolution of small Tae1 proteins. Relevant protein marker should be indicated. In addition, why there is no western blot analysis of C30A variant?
    4. It is exciting that a hyperactive Tae1 C110S variant is also naturally encoded in a subset of Proteobacteria outside of P. aeruginosa. The authors showed higher binding affinity of WT Tae1 than C110S Tae1, which correlated with lower fitness of C110S variant in a competition setup (Fig. 6C, 6E). The authors suggest that "Tae1 of C110S variant lyses kin cells at a faster rate than Tae1 WT can bind and inhibit killing, leading to a fitness cost for this strain" (Line 460-463). To claim this, expression levels of endogenous Tae1 of both WT and C110S should be shown as well as their secretion levels to rule out the effect of protein abundance and secretion levels may affect the fitness. It would be also recommended to set up a real interbacterial competition assay by selecting the survival cfu of prey cells.

    Minor comments:

    1. Is Tae1 previously named as Tse1? Please clarify and indicate the previous name and accession number. As stated in Line 61" Although many T6S bacteria deploy similar toxins, interbacterial outcomes can vary considerably depending on the bacterial species engaged in T6S-mediated competition", the authors should also cite other relevant references showing differential Tae toxicity from different organisms (such as Serratia marcescens. Ssp1 and Ssp2 from English et al., 2012, Enterobacter cloacae Tae4 from Zhang et al., 2012, and Agrobacterium tumefaciens Tae from Yu et al., 2021). The manuscript shall gain more insights by discussing the biological significance of their conservation yet distinct toxicity and potential condition-specific activity of Tae toxins studied in different bacterial lineage besides those in P. aeruginosa.
    2. Line 111-113 "the Tae1 protein from P. aeruginosa, which is injected into E. coli and leads to cell lysis": citations are needed here.
    3. The heatmap in Fig. 4E also include those with mixed phenotypes. Are the averaged fitness score meaningful since some residues are likely derived from the mixed phenotypes, which make the data less reliable. I suggest the authors to only include those true GOF or indicate which one is true GOF and which one is from mixed phenotypes.

    Significance

    This manuscript used innovative approaches to investigate the mechanism and biological significance underlying the differential toxicity of the bacterial T6SS amidase effector (Tae). Tae superfamily can be classified into four families (Tae1-4), which are universally encoded in T6SS of diverse Proteobacteria. It is intriguing that Tae toxins classified in the same family produced by different bacterial lineage/strains exhibit distinct lysis/antibacterial activity but the underlying mechanism is unknown. This manuscript provided evidence suggesting the existing natural diversity of Tae1 in substrate-specificity and toxin-immunity interactions, which are the key selective pressures for shaping diversity across the Tae1 toxin superfamily. The findings provide an explanation how bacterial toxin effectors evolve in the context of interbacterial antagonism, which have not been answered from previous literatures (Russell et al., 2012; English et al., 2012; Chou et al., 2012; Zhang et al., 2013; Yu et al., 2021). The methods combining deep mutagenesis scan, biochemical, and structural analysis provide a comprehensive and unbiased view to understand the diversity of Tae1 family and their corresponding phenotypes and biochemical features. As a molecular microbiologist working on bacterial secretion systems and their effectors not familiar with structural studies, I am better qualified in evaluating the biological and biochemical data but not the structural studies in NMR and structural modeling. However, I highly appreciate the authors who nicely presented the story by explaining the concept of each method which allows the reviewers/readers to understand the data even though not within their expertise.

    Russell et al., Cell Host Microbe 11:538-549. https://doi.org/10.1016/j.chom.2012.04.007 English et al., Mol Microbiol 86:921-936. https://doi.org/10.1111/mmi.12028. Chou, S. et al. Cell Reports 1, 656-664. DOI: 10.1016/j.celrep.2012.05.016 Zhang et al., J Biol Chem 288:5928-5939. https://doi.org/10.1074/jbc.M112.434357 Yu et al., J Bacteriol 203:e00490-20. https://doi.org/10.1128/JB.00490-20.

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    Referee #1

    Evidence, reproducibility and clarity

    In this paper the authors characterize a member of the bacterial T6SS amidase effector (Tae) superfamily of toxins that are delivered by the Type 6 Secretion system of Pseudomonas aeruginosa into target prey bacterial cells. The authors focused on why this toxic effector because it shows different potency when delivered to different target species despite the fact that all target species have peptidoglycan, the substrate that Tae attacks. The authors use powerful approaches such as deep mutational scanning (DMS), to define critical residues near the Tae active site and other sites that affect its enzymatic activity and interaction with its cognate immunity protein. The discovery of the C110S mutation which increases Tae activity is a fine example of the power of this approach. When combined with structural biological analysis, the results of the study and discussion in the manuscript is of broad interest to the community of scientists interested in toxic bacterial effectors that digest the cell wall and also others that are interested in the remodeling of peptidoglycan during cell growth and shape determination. I would recommend acceptance of this paper for publication after the authors address a few minor comments:

    1. The authors observe PG changes caused by D-met (Fig 4 and relevant text). I'm curious as to whether changes in lysis are caused by differences in PG crosslinking or PG density. They point out that the sugar binding surface of WT could localize the PG digestion (paragraph at line 521) which would no longer be required at lower density PG. However, my concern is that they propose that variation in Tae activity in different target organisms could be explained by differences in PG affinity without testing this DMS screen in any other strain, let alone species.
    2. They also don't screen any Tae1 homologs, though they address one residue in their phylogeny. I'm not sure if there are species with such a low-density PG layer, so their repeated connection to Tae's variable lytic capacity between species in the text and discussion seems tenuous until they do a DMS screen with their plasmid library in another species (or at least another strain).
    3. If WT Tae1 has some checking mechanism to ensure it's in the PG layer, I can imagine it might be slower to fire in less dense PG. That would also make sense given the chemical perturbations in Fig 3 where residues on the opposite face from the catalytic site are involved in binding PG. I would be interested to see if WT Tae1 can bind multiple PG chains or binds at higher affinity. A calorimetry approach like the one they use later may answer those questions, but that might be outside the range of this paper.
    4. It's also worth looking around for E. coli PG synthesis mutants that don't change the PG polymer structure, only the density. That might also happen if the bugs are grown under osmotic stress, which should at the very least stretch out the sacculus. That may help differentiate differences in PG composition from differences in chain density. Perhaps subinhibitory amounts of drugs that affect PG synthesis my have the same of effect of increasing or even decreasing Tae potency by modulating PG density. Of course, this may have to be done under protective osmotic conditions. Have the authors tried these sorts of experiments and if so, please comment on the trends even if the data will not be presented in this report.

    Significance

    When combined with structural biological analysis, the results of the study and discussion in the manuscript is of broad interest to the community of scientists interested in toxic bacterial effectors that digest the cell wall and also others that are interested in the remodeling of peptidoglycan during cell growth and shape determination.

  6. Version published to 10.1101/2022.02.15.480620v2 on bioRxiv
  7. Version published to 10.1101/2022.02.15.480620v1 on bioRxiv