Reassessing the substrate specificities of the major Staphylococcus aureus peptidoglycan hydrolases lysostaphin and LytM

  1. Lina Antenucci
  2. Salla Virtanen
  3. Chandan Thapa
  4. Minne Jartti
  5. Ilona Pitkänen
  6. Helena Tossavainen
  7. Perttu Permi

  • Curated by eLife

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    eLife assessment

    This manuscript describes a valuable study aimed at identifying the substrate specificity of two cell wall hydrolases LSS and LytM in S. aureus. The authors show that LytM has a novel function of cleaving D-Ala-Gly instead of only Gly-Gly by using synthetic substrates and compelling NMR-based real-time kinetics measurements.

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Abstract

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well-understood, the function, regulation, and mechanism of action of PG hydrolases characterized as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined.In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

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  1. eLife

    eLife assessment

    This manuscript describes a valuable study aimed at identifying the substrate specificity of two cell wall hydrolases LSS and LytM in S. aureus. The authors show that LytM has a novel function of cleaving D-Ala-Gly instead of only Gly-Gly by using synthetic substrates and compelling NMR-based real-time kinetics measurements.

  2. eLife

    Reviewer #1 (Public Review):

    Summary:

    The manuscript aimed at elucidating the substrate specificity of two M23 endopeptidase Lysostaphin (LSS) and LytM in S. aureus. Endopeptidases are known to cleave the glycine-bridges of staphylococcal cell wall peptidoglycan (PG). To address this question, various glycine-bridge peptides were synthesized as substrates, the catalytic domain of LSS and LytM were recombinantly expressed and purified, and the reactions were analyzed using solution-state NMR. The major finding is that LytM is not only a Gly-Gly endopeptidase, but also cleaves D-Ala-Gly. Technically, the advantage of using real-time NMR was emphasized in the manuscript. The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM. However, the biological significance and relevance of the conclusions remain clear, as the results are mostly from synthetic substrates.

    Strengths:

    The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM.

    Comments on the revised version:

    The authors have addressed most of my concerns. I agree that the physiological functions of LytM are not in the scope of the current study.

  3. eLife

    Reviewer #2 (Public Review):

    Summary:

    This work investigates the enzymatic properties of lysostaphin (LSS) and LytM, two enzymes produced by Staphylococcus aureus and previously described as glycyl-glycyl endopeptidases. The authors use synthetic peptide substrates mimicking peptidoglycan fragments to determine the substrate specificity of both enzymes and identify the bonds they cleave.

    Strengths:

    - This work is addressing a real gap in our knowledge since very little information is available about the substrate specificity of peptidoglycan hydrolases.
    - The experimental strategy and its implementation are robust and provide a thorough analysis of LSS and LytM enzymatic activities. The results are very convincing and demonstrate that the enzymatic properties of the model enzymes studied need to be revisited.
    - I think the experimental work is extremely well designed and way beyond what people usually do in the field. I believe that this sets a precedent that is worth acknowledging.

  4. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Reviewer #1 (Recommendations For The Authors):

    (1) Figure 2B and 2D: unlike what is written in the results part, the results are not consistent, but opposite: LSS has higher activity in 2B, less in 2D.

    The activities in Figure 2B come from NMR kinetic experiments with pGly, whereas Figure 2D reports on activity towards whole S. aureus cells. The LytM and LSS activities in these two experiments are indeed not directly comparable, but served to highlight the fact that simple pentaglycine is a poor model substrate for M23 enzymes. We carried out a turbidity assay with pristine enzymatic preparation and indeed it is highly consistent both with the kinetic assay using pentaglycine (Fig. 2B) as well as with larger PG fragments (Fig. 2K) indicating that the catalytic domain of LSS is significantly more efficient than LytM in hydrolyzing cells from community acquired methicillin resistant S. aureus strain USA300 as well as synthetic PG fragments. The corresponding paragraph in Results has now been updated and rephrased.

    (2) Figure 2, panel K missing statistical analysis, which makes it difficult to appreciate if the difference is significant. If it is a one-time experiment or a single value, the value should be presented as a table. The corresponding text in the results part is confusing. The fold change or drop in percentage is unclear in the figure.

    We have added a table (panel L) to Figure 2, which shows absolute values of LSS and LytM hydrolysis rates. Indeed, most of the values are from single NMR kinetic measurements, however, PG fragment (2) for LSS and PG fragment (3) for LytM were measured as duplicates to verify the reproducibility of the data. This is now mentioned in Figure 3 legend and in the Materials and Methods. Also, the corresponding text in the Results has been updated and rephrased.

    (3) Figure 3H: the cleavage of D-ala-gly is unclear, the cleavage products need to be labeled and quantified. The experiment used purified PG treated with mutanolysin. Presumably, mixed monomers, dimers, trimers, and multimers are used. It would be helpful to show the HPLC profile of the purified muropeptide. It would be informative to analyze which fractions generate D-ala-gly. In addition, the intact murein sacculus should be included.

    For the sake of clarity, we have moved the 13C-HMBC spectra presented in Figure 3H to Fig. S7 in the Supplementary Material. The full carbonyl carbon region of the reference (prior to addition of enzyme) 13C-HMBC spectrum together with larger expansions of spectra acquired from enzyme-treated muropeptides are now shown. Furthermore, graphical presentations of identified PG fragments due to LSS/LytM activity are included. No HPLC analysis of the muropeptides was performed at this stage. Being insoluble, the intact murein sacculus is not amenable to liquid-state NMR studies, but we envisage studies of this remarkably complex structure also with solid-state NMR.

    Reviewer #2 (Recommendations For The Authors):

    Overall, the experiments address the question asked by the authors and no additional experiments are required to strengthen the conclusions drawn.

    Abstract:

    The abstract is not well written and more specific (and accurate) information should be provided by the authors.

    We are grateful for the constructive and helpful comments to improve our manuscript. The abstract has now been modified by taking into account the Reviewer’s suggestions.

    Introduction

    The intro is relatively long and wordy. It could most certainly be shortened and written in a more simple way to make it more impactful.

    The introduction has now been modified by taking into account the Reviewer’s suggestions.

    (2) One of the peptide stems in Figure 1 is missing a pentaglycine side chain; I would recommend increasing the font size; the peptide stem looks like it is attached to the carbon in position 2, it may be a good idea to move it to the left?

    We thank the Reviewer for this comment. Figure 1 has been improved, the frameshift has been fixed and the non-cross-linked pGly bridge has been included to the lysine side-chain in tetraStem.

    Results

    Figure 2 is a bit overwhelming and its description is sketchy. Fig 2B shows a much higher activity of LSS on pGly as compared to LytM whilst 2K shows a very similar rate.

    We have rearranged Figures 1 and 2 by moving the original panel J in Figure 2 (structures of PG fragments) to Figure 1 panel C. The bar graph in Figure 2J now shows absolute rates of substrate hydrolysis for 2 mM LSS and LytM. These indicate that LSS is much more efficient against PG fragments in vitro in comparison to LytM. Rates normalized with respect to pGly are shown in Figure 2K. Also, a table showing absolute rates of hydrolysis for 2 mM LSS and 50 mM LytM has been included in Figure 2, panel L. In this Table, the values for PG fragments 2 and 3 were determined by two independent measurements to test and accredit the reproducibility of the method. This is also now elaborated further in the Materials and Methods.

    Figure 3 is impressive and very informative but again hard to follow.

    - Panels 3A and 3B are nicely conceived but the resolution is rather poor and it is difficult to know exactly where the arrows point.

    We very much value suggestions given by the Reviewer to improve readability of our manuscript. In the case of Figure 3, we have now greatly enhanced the resolution and readability of the figure by horizontal scaling of panels A and B.

    Figure 4 shows a comparative analysis of catalytic rate using various substrates, the authors may want to present graphs with the same y-axis to get the most out of the comparison between substrates.

    The scaling of the y-axis is the same for all the substrates now. In addition, we have reorganized the panels in the figure to enhance readability.

    Figure 5: - The same remark as above, please cite all panels in alphabetical order.

    Citing to Figure 5 has now been revised.

    Material and methods:

    - How were the peptide concentrations determined? It may be useful to indicate if specific conditions were required to solubilize some peptides, pGly is particularly insoluble in aqueous solutions.

    - Page 19, replace cpm by rpm; biological or technical replicates?

    These have now been added and edited accordingly.

  5. Version published to 10.7554/elife.93673.2 on eLife
  6. Version published to 10.7554/elife.93673 on eLife
  7. Version published to 10.1101/2023.10.13.562287v2 on bioRxiv
  8. Version published to 10.7554/elife.93673.1 on eLife
  9. eLife

    Author Response

    Public Reviews:

    Reviewer #1 (Public Review):

    Summary:

    The manuscript aimed at elucidating the substrate specificity of two M23 endopeptidase Lysostaphin (LSS) and LytM in S. aureus. Endopeptidases are known to cleave the glycine-bridges of staphylococcal cell wall peptidoglycan (PG). To address this question, various glycine-bridge peptides were synthesized as substrates, the catalytic domain of LSS and LytM were recombinantly expressed and purified, and the reactions were analyzed using solution-state NMR. The major finding is that LytM is not only a Gly-Gly endopeptidase, but also cleaves D-Ala-Gly. Technically, the advantage of using real-time NMR was emphasized in the manuscript. The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM. However, the biological significance and relevance of the conclusions remain clear, as the results are mostly from synthetic substrates.

    Strengths:

    The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM.

    Weaknesses:

    1. Significance: while the current study provided a detailed analysis of various substrates, the conclusions are mainly based on synthesized peptides. One experiment used purified muropeptides (Fig. 3H); however, the results were unclear from this figure.

    We acknowledge the Reviewer for comments and concerns regarding the potential weaknesses of this study.

    Because peptidoglycan is insoluble, as such it is not amenable to solution-state NMR studies. However, soluble peptidoglycan (PG) fragments for NMR analyses can be obtained by digesting bacterial sacculi or via chemical synthesis. Whereas digestion results in mixtures of products, synthesis yields pure molecules. Analysis of NMR spectra of muropeptide-mimicking synthetic peptides before and after enzyme addition provides tools to identify peaks in the much more complex spectra of mutanolysin-treated sacculus.

    We will improve data presentation in Figure 3H in the revised version of our manuscript and emphasize the similarity of product peaks in spectra acquired from experiments using either synthetic peptides or mutanolysin-digested sacculus.

    The results from synthesized peptides may not necessarily correlate with their biological functions in vivo.

    The Reviewer refers several times to the use of synthetic peptides in this study. While it is unclear to us whether the concern is about the synthetic nature of the molecules or because the peptides are devoid of PG disaccharide units, it is true that PG fragments lack the 3D architecture present in intact sacculus, and thus cannot perfectly mimic the in vivo milieu. The fragments, as well as purified sacculus, also lack all other components present in an intact bacterial cell wall. Our largest synthetic peptide (7), however, represents a crosslinked muropeptide (stem-pentaGly-stem) which according to the structural model recently presented by Razew et al. (2023) (Staphylococcus aureus sacculus mediates activities of M23 hydrolases. Nat Commun 14, 6706) is large enough to cover the peptidic interaction interface between substrate and enzyme.

    Secondly, the study used only the catalytic domain of both proteins. It is known that the substrate specificity of these enzymes is regulated by their substrate-binding domains. There is no mention of other domains in the manuscript and no justification of why only the catalytic domain was studied. In short, the relevance of the results from the current study to the enzymes' actual physiological functions remains to be addressed, which attenuated the significance of the study.

    Lysostaphin catalytic domain was used for experimental simplicity and to allow direct comparison with LytM catalytic domain. Because lysostaphin cell-wall targeting (SH3b) domain interacts with the substrate with variable affinities depending on the substrate structure (Tossavainen et al., Structural and functional insights into lysostaphin-substrate interaction, Front. Mol. Biosci. 5, 60 (2018) and Gonzalez-Delgado et al., Two-site recognition of Staphylococcus aureus peptidoglycan by lysostaphin SH3b, Nat. Chem. Biol. 16, 24-30 (2020)), we would have had skewed results on kinetics because of this interaction.

    Catalytic domains were used also in the article by Razew et al. (Staphylococcus aureus sacculus mediates activities of M23 hydrolases. Nat Commun 14, 6706 (2023)). They showed that mature lysostaphin and lysostaphin catalytic domain hydrolysed the same Gly-Gly bonds.

    Moreover, full-length LytM is catalytically inactive. This is because the linker between its N-terminal and catalytic domains occludes the catalytic site (Odintsov et al. Latent LytM at 1.3 Å resolution. J. Mol. Biol. 225, 775 (2004)). LytM catalytic domain without its N-terminal segment is active (Odintsov et al (2004) and Firczuk et al. Crystal structure of active LytM. J. Mol. Biol 354, 578 (2005)).

    1. Impact and novelty:

    (1) the current study provided evidence suggesting the novel function of LytM in cleaving D-Ala-Gly. The impact of this finding is unclear. The manuscript discussed Enterococcus faecalis EnpA. But how about other M23 endopeptidases? What is biological relevance?

    EnpA was specifically mentioned because it has been reported to also cleave the D-Ala-Gly bond. Structural similarities between the enzymes could reveal the basis for this bond specificity. Moreover, the focus of the study was not to reveal the biological function of LytM but rather to understand which amino acid substitutions lead to differences in specificities in the two structurally very similar enzymes.

    (2) A very similar study published recently showed that the activity of LSS and LytM is regulated by PG cross-linking: LSS cleaves more cross-linked PG and LytM cleaves less cross-linked PG (Razew, A., Laguri, C., Vallet, A., et al. Staphylococcus aureus sacculus mediates activities of M23 hydrolases. Nat Commun 14, 6706 (2023). The results of this paper are different from the current study whereby both LSS and LytM prefer cross-linked substrates (Fig, 2JKL). Moreover, no D-Ala-Gly cleavage was observed by LytM using purified PG substrate from Razew A et al. An explanation of inconsistent results is needed here. In my opinion, the knowledge generated from the current study has not been fully settled. If the results can be validated, the contribution to the field is incremental, but not substantial.

    Another point raised by the Reviewer concerned the inconsistent results between our study and the recent paper by Razew et al. (2023) regarding LytM D-Ala--Gly cleavage. The explanation might lie in the type of NMR data acquired and its interpretation. We identified all hydrolysis products using 1H, 13C multiple bond correlation NMR spectra acquired from samples dissolved in deuterated buffers. Use of C-H signals is advantageous in that they are not prone to chemical exchange phenomena and enable unambiguous chemical shift assignment. Based on shown NMR spectra, Razew and co-workers identified cleaved muropeptide bonds by observing product glycine peaks in 1H, 15N correlation spectra, specifically amide peaks of product C-terminal glycines appearing in the 114-117 ppm 15N region of spectra of samples treated with LytM/LSS. D-Ala--Gly cleavage, however produces an N-terminal glycine, whose signal due to chemical exchange is not typically observed in regular N,H correlation spectra. Razew and co-workers validated their observations with UPLC-MS analysis. However, to our understanding, their data analysis was based on the assumption that LytM cleaves between Gly4-Gly5 (or Gly1-Gly2 using our numbering), and accordingly only masses corresponding to potential products containing 1 to 4 glycines anchored to the lysine side chain were considered.

    (3) The authors emphasized a few times in the text that it is superior to use NMR technology. In my opinion, NMR has certain advantages, such as measuring the efficacy of cleavage, but it is not that superior. It should be complementary to other methods such as mass spectrometry. In addition, more relevant solid-state NMR using intact PG or bacterial cells was not discussed in the study. I am of the opinion that the corresponding text should be revised.

    We value and agree with the Reviewer’s opinion that NMR spectroscopy is complementary to other methods e.g., mass spectrometry. However, in this particular case, NMR provided simultaneously information on reaction kinetics as well as scissile bonds in the substrates, which allowed us to compare rates of hydrolysis in different PG fragments and reshape the substrate specificities of LytM/LSS. We also agree that solid-state NMR is a wonderful technique. In our revised manuscript, we will edit the text accordingly.

    1. The conclusions are not fully supported by the data

    As mentioned above, the conclusions from synthesized peptide substrates may not necessarily reveal physiological functions. The conclusions need to be validated by more physiological substrates.

    As pointed out above in our response to the potential weaknesses of this study, the aim of this work was not to reveal the physiological function of LytM but to glean information on its substrate specificity that echoes its functional role in a substrate level. Hitherto LytM has been shown to cleave amide bonds between glycines without providing detailed information about the specific scissile bonds in the established PG components in S. aureus cell wall. The same holds true for lysostaphin as well. This study provides concomitantly information on the rates of hydrolysis and scissile bonds of these two enzymes. We deduced that LytM, and especially lysostaphin substrate specificity is defined by D-Ala-Gly cross-linking, which is a structural property, whereas Razew et al. (2023) discuss about “more cross-linked” and “less cross-linked PG”, which is a supramolecular asset or density.

    1. There are some issues with the presentation of the figures, text, and formatting.

    We are grateful to the Reviewer for bringing up issues in figures and text. We will address these in the revised version of the manuscript.

    Reviewer #2 (Public Review):

    Summary:

    This work investigates the enzymatic properties of lysostaphin (LSS) and LytM, two enzymes produced by Staphylococcus aureus and previously described as glycyl-glycyl endopeptidases. The authors use synthetic peptide substrates mimicking peptidoglycan fragments to determine the substrate specificity of both enzymes and identify the bonds they cleave.

    Strengths:

    • This work is addressing a real gap in our knowledge since very little information is available about the substrate specificity of peptidoglycan hydrolases.
    • The experimental strategy and its implementation are robust and provide a thorough analysis of LSS and LytM enzymatic activities. The results are very convincing and demonstrate that the enzymatic properties of the model enzymes studied need to be revisited.

    Weaknesses:

    • The manuscript is difficult to read in places and some figures are not always presented in a way that is easy to follow. This being said, the authors have made a good effort to present their experiments in an engaging manner. Some recommendations have been made to improve the current manuscript but these remain minor issues.

    We thank the Reviewer for providing positive feedback on our manuscript and for appreciating the systematic work behind this study which aims to unknot the substrate specificity of two S. aureus PG hydrolyzing enzymes. We are grateful for the comments aiming to improve the presentation of the current version of manuscript and we will take these into account while preparing the revised version of the manuscript.

  10. eLife

    eLife assessment

    This manuscript describes a valuable study aimed at identifying the substrate specificity of two cell wall hydrolases LSS and LytM in S. aureus. The authors show that LytM has a novel function of cleaving D-Ala-Gly instead of only Gly-Gly by using synthetic substrates and convincing NMR-based real-time kinetics measurements. The biological relevance of the reported results will have to be investigated in future in vivo experiments.

  11. eLife

    Reviewer #1 (Public Review):

    Summary:
    The manuscript aimed at elucidating the substrate specificity of two M23 endopeptidase Lysostaphin (LSS) and LytM in S. aureus. Endopeptidases are known to cleave the glycine-bridges of staphylococcal cell wall peptidoglycan (PG). To address this question, various glycine-bridge peptides were synthesized as substrates, the catalytic domain of LSS and LytM were recombinantly expressed and purified, and the reactions were analyzed using solution-state NMR. The major finding is that LytM is not only a Gly-Gly endopeptidase, but also cleaves D-Ala-Gly. Technically, the advantage of using real-time NMR was emphasized in the manuscript. The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM. However, the biological significance and relevance of the conclusions remain clear, as the results are mostly from synthetic substrates.

    Strengths:
    The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM.

    Weaknesses:
    1. Significance: while the current study provided a detailed analysis of various substrates, the conclusions are mainly based on synthesized peptides. One experiment used purified muropeptides (Fig. 3H); however, the results were unclear from this figure. The results from synthesized peptides may not necessarily correlate with their biological functions in vivo. Secondly, the study used only the catalytic domain of both proteins. It is known that the substrate specificity of these enzymes is regulated by their substrate-binding domains. There is no mention of other domains in the manuscript and no justification of why only the catalytic domain was studied. In short, the relevance of the results from the current study to the enzymes' actual physiological functions remains to be addressed, which attenuated the significance of the study.

    2. Impact and novelty: (1) the current study provided evidence suggesting the novel function of LytM in cleaving D-Ala-Gly. The impact of this finding is unclear. The manuscript discussed Enterococcus faecalis EnpA. But how about other M23 endopeptidases? What is biological relevance? (2) A very similar study published recently showed that the activity of LSS and LytM is regulated by PG cross-linking: LSS cleaves more cross-linked PG and LytM cleaves less cross-linked PG (Razew, A., Laguri, C., Vallet, A., et al. Staphylococcus aureus sacculus mediates activities of M23 hydrolases. Nat Commun 14, 6706 (2023). The results of this paper are different from the current study whereby both LSS and LytM prefer cross-linked substrates (Fig, 2JKL). Moreover, no D-Ala-Gly cleavage was observed by LytM using purified PG substrate from Razew A et al. An explanation of inconsistent results is needed here. In my opinion, the knowledge generated from the current study has not been fully settled. If the results can be validated, the contribution to the field is incremental, but not substantial. (3) The authors emphasized a few times in the text that it is superior to use NMR technology. In my opinion, NMR has certain advantages, such as measuring the efficacy of cleavage, but it is not that superior. It should be complementary to other methods such as mass spectrometry. In addition, more relevant solid-state NMR using intact PG or bacterial cells was not discussed in the study. I am of the opinion that the corresponding text should be revised.

    3. The conclusions are not fully supported by the data
    As mentioned above, the conclusions from synthesized peptide substrates may not necessarily reveal physiological functions. The conclusions need to be validated by more physiological substrates.

    4. There are some issues with the presentation of the figures, text, and formatting.

  12. eLife

    Reviewer #2 (Public Review):

    Summary:
    This work investigates the enzymatic properties of lysostaphin (LSS) and LytM, two enzymes produced by Staphylococcus aureus and previously described as glycyl-glycyl endopeptidases. The authors use synthetic peptide substrates mimicking peptidoglycan fragments to determine the substrate specificity of both enzymes and identify the bonds they cleave.

    Strengths:
    - This work is addressing a real gap in our knowledge since very little information is available about the substrate specificity of peptidoglycan hydrolases.
    - The experimental strategy and its implementation are robust and provide a thorough analysis of LSS and LytM enzymatic activities. The results are very convincing and demonstrate that the enzymatic properties of the model enzymes studied need to be revisited.

    Weaknesses:
    - The manuscript is difficult to read in places and some figures are not always presented in a way that is easy to follow. This being said, the authors have made a good effort to present their experiments in an engaging manner. Some recommendations have been made to improve the current manuscript but these remain minor issues.

  13. Version published to 10.1101/2023.10.13.562287v1 on bioRxiv