From the journal RSC Chemical Biology Peer review history

Berlin, June 9, 2020

Dear Dr Wagner:

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

Manuscript ID: CB-ART-05-2020-000060
TITLE: Antibacterial Activity of a Dual Peptide Targeting the Escherichia coli Sliding Clamp and the Ribosome


After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

************

EDITORS COMMENT:
The legend text of Table 2 ([a] and [b]) is not visible. Particularly, for the peptides of the Onc-series it would be highly desirable to determine the MBC values!

Reviewer 1

The paper by André et al. reports the design, synthesis and evaluation of peptides designed by fusing a proline-rich antimicrobial peptide (PrAMP), which can easily enter e. coli cells and target the 70S ribosome, with peptides targeting the sliding clamp (SC), which were developed by the authors in a previous study. They first overexpress their SC peptides intracellularly to demonstrate that it can kill cells, and then synthesize and test fusion peptides with PrAMPs, including FITC labeled analogs, for binding to the respective targets by ITC (SC and 70S ribosome), and for antibacterial activity in culture and in an fly infection model. The study is nicely designed, complete, well written and interesting to read, and has originality. The experimental part is clear and complete, and the SI provides the necessary additional characterization data. The key conclusion of the study is that it is possible to obtain a bactericidal effect by engaging SC, as evidenced by the activity of the FITC labeled fusion peptides, which are cell penetrating but lost their 70S ribosome blocking activity, while the same peptides with a scrambled sequence for the SC targeting part are inactive. I recommend publishing the paper after consideration of the following comments:

1) the discussion on page 10 left column about the fact that the abundance of the 70S ribosome might hijack the peptide and not allow targeting SC is interesting, but the explanation could be made clearer. By my own calculation 6000 copies per cell (the lower end) would give about 10 micromolar concentration (27 mg/mL) of the 70S ribosome in the cell (assuming 1 fL per cell). With a nanomolar affinity, one would expect that the cells accumulate the peptide, otherwise it’s not possible to have and MIC at 0.2 microM. The accumulation inside the cells could result in more activity against other targets (here SC), rather than hiding the peptide away, unless the localization is different. For future studies, the authors could think of finding a FITC labeled peptide which would still be active against the ribosome (it should not be that hard: just label the C-terminus) to do localization studies inside cells: perhaps the peptides colocalizes with the ribosome.

2) A comment must be added in the intro and/or conclusion on the relevance of SC as a target for bacteria other than E. coli. A new antibiotic does not make sense if it’s only for E. coli. The issue of resistance should also be commented, if not tested.

Reviewer 2

The manuscript by Burnouf, Guichard, Wagner and coworkers describes the development and evaluation of antibacterial peptides that target the sliding clamp (SC) and the ribosome. The study is thoroughly performed and the manuscript is very well laid out and clearly communicated.
With the growing importance for developing alternative strategies to target resistant bacteria, i highly recommend acceptance of this excellent study after minor revision noted below.

Minor points:
1. Table 2 appears to have its caption/footnotes missing.
2. Neither in Fig 3 nor by the full images shown in the Supplementary Information could I find mentioned how many times this experiment were repeated. At least two biological replicates should be performed.
3. HR-MS analyses should be provided for the synthesised peptides.

We wish to thank the editor and the reviewers for their positive and constructive analyses of our work.
All the modifications made within the main text are highlighted in yellow (as in the marked revised version).
Bellow are our answers to the Editor and Reviewers comments.

EDITORS COMMENT:
The legend text of Table 2 ([a] and [b]) is not visible. Particularly, for the peptides of the Onc-series it would be highly desirable to determine the MBC values!

Answer:
We are sorry about the lack of a legend to Table 2. We have added the missing legend in the revised version of the manuscript as follow:
" Ampicillin and tetracycline antibiotics were included in the assays as positive controls. n.d.: not determined. [a] Ranges of Minimal Inhibitory Concentrations determined in a minimum of 2 independent biological replicates are indicated. [b] Minimal Bactericidal Concentrations determined in a minimum of 2 independent biological replicates are indicated. See the Experimental section for detailed procedures."

We have determined the MBC values for the peptides of the Onc-series (MBC values for Onc112 is 1 µM; Onc112-P7: 2 µM; Onc112-P7Scr: 2µM) and for the Py-P7Scr peptide (MBC: 16 µM) and have added the obtained values within the new Table 2. During these experiments we observed that the MIC for the Py-P7Scr peptide can reach 16 µg/ml. We have modified the Table 2 and the main text accordingly:

Within the text, page 8, left column: " Minimal Bactericidal Concentration (MBC) determination for Py and Onc derived peptides confirms the bactericidal effect of these peptides, which appear to be 1 to 16 times more potent than ampicillin (Table 2)."

REVIEWER REPORT(S):
Referee: 1

Comments to the Author
The paper by André et al. reports the design, synthesis and evaluation of peptides designed by fusing a proline-rich antimicrobial peptide (PrAMP), which can easily enter e. coli cells and target the 70S ribosome, with peptides targeting the sliding clamp (SC), which were developed by the authors in a previous study. They first overexpress their SC peptides intracellularly to demonstrate that it can kill cells, and then synthesize and test fusion peptides with PrAMPs, including FITC labeled analogs, for binding to the respective targets by ITC (SC and 70S ribosome), and for antibacterial activity in culture and in an fly infection model. The study is nicely designed, complete, well written and interesting to read, and has originality. The experimental part is clear and complete, and the SI provides the necessary additional characterization data. The key conclusion of the study is that it is possible to obtain a bactericidal effect by engaging SC, as evidenced by the activity of the FITC labeled fusion peptides, which are cell penetrating but lost their 70S ribosome blocking activity, while the same peptides with a scrambled sequence for the SC targeting part are inactive. I recommend publishing the paper after consideration of the following comments:

1) the discussion on page 10 left column about the fact that the abundance of the 70S ribosome might hijack the peptide and not allow targeting SC is interesting, but the explanation could be made clearer. By my own calculation 6000 copies per cell (the lower end) would give about 10 micromolar concentration (27 mg/mL) of the 70S ribosome in the cell (assuming 1 fL per cell). With a nanomolar affinity, one would expect that the cells accumulate the peptide, otherwise it’s not possible to have and MIC at 0.2 microM. The accumulation inside the cells could result in more activity against other targets (here SC), rather than hiding the peptide away, unless the localization is different. For future studies, the authors could think of finding a FITC labeled peptide which would still be active against the ribosome (it should not be that hard: just label the C-terminus) to do localization studies inside cells: perhaps the peptides colocalizes with the ribosome.

Answer:
We totally agree with the referee 1's calculations and that the necessary accumulation of the peptides in the cell may result in more activity against other targets. The point is that we can't observed a clear difference in MIC between the PrAMP-P7 and the PrAMP-P7Scr peptides in vitro (Table 2) and this observation, coupled to the known activity of the FITC-Py-P7 peptide (that does not interact with the ribosome), lead us to elaborate the discussion on the possible hijacking of the peptides by the ribosome (discussion on page 8, left column). Another way to express this hypothesis is that due to the relative targets abundance and the respective affinities of the peptides for each of them, calculation predict that the ribosomal target will be the first to be sufficiently engaged to trigger a toxic effect. In such a case, we will not be abble to detect a "secondary" toxic effect mediated by the SC binding sequence (you can't be more dead than dead...).
We are actually thinking about a way to determine wether the bifunctional peptides are abble to bound both targets simultaneously, in vitro and in vivo.
For what concern the discussion on the clearly observed superior activity of the Onc-P7 peptide in vivo as compared to Onc and Onc-P7Scr (discussion on page 10, left column) we must rely on some parameters that are modified in the in vivo assays. We think that we provide plausible explanations to account for this in vitro/in vivo discrepency.


2) A comment must be added in the intro and/or conclusion on the relevance of SC as a target for bacteria other than E. coli. A new antibiotic does not make sense if it’s only for E. coli.

Answer:
To clearly address this important point, we added the following sentence within the "Concusions", page 10:
" Moreover, this bactericidal activity leads to a protective effect in living animals challenged by a bacterial infection. We previously determined the high degree of conservation of key residues involved in the binding of short peptides within the SC binding pocket among 128 bacteria from various phylae8. Moreover, there is a strict conservation of the SC binding pocket between E. coli and other human pathogens such as K. pneumoniae, P. aeruginosa and A. Baumanii (among others), all identified by OMS as critical priority strains. Collectivelly, these results further validate SC as a molecular target for the development of a new class of antibiotics."

2)...The issue of resistance should also be commented, if not tested.
We think that the issue of resistance is already clearly commented in the "Introduction" and "Conclusions" parts:
" Ideally, these new drugs should also limit the frequency of resistance emergence. In this context, the bacterial replication processivity factor, or sliding clamp (SC) is a promising target because of its central function in the bacterial replisome dynamics2–4. This protein (DnaN) forms a homodimeric molecular hub, which interacts with many partners involved in bacterial DNA metabolism. In particular, all DNA polymerases interact with SC5 via a short peptide motif, for which a consensus sequence (QL[S/D]LF) has been defined6, and the hydrophobic pocket where these peptides interact7,8 has been recognized as a potential molecular target for new drug development4. It is anticipated that blocking the interaction between SC and the replicative polymerase will lead to cell death. More interestingly, as the specialized, SOS-regulated DNA polymerases (PolII, IV and V) are required for mutagenesis, inhibition of their activities should limit the possibility for bacteria to develop new resistance. Actually, these polymerases have been shown to directly participate in the establishment of ciprofloxacin resistance in E. coli9 and the necessity to interact with the SC to perform efficient mutagenesis has been demonstrated5,10."
" By its involvement in DNA replication and mutagenesis and its yet unexploited potential as antimicrobial target, the bacterial SC represents a chance to develop antibiotics that will contribute to the fight against antimicrobial resistance9,50. Moreover, targeting simultaneously two distinct and essential metabolic pathways mathematically reduce the probability of resistance acquisition."
We totally agree that directly testing the influence of the exposition of bacteria to SC binding peptides onto mutation frequency and resistance acquisition is an important point. This will be the subject of a dedicated future study with specifically designed peptides. We however added the following sentence within the "Conclusions" (page 10) to highlight the real potential of such new antibiotics in the fight against resistance:
" Remarkably, Kling and coworkers49 showed that resistance of Mycobacterium smegmatis to griselimycin occured at the extremely low frequency of 5 x 10-10 highlighting thus the potential of SC binding molecules to reduce the rising of antimicrobial resistance."



Referee: 2

Comments to the Author
The manuscript by Burnouf, Guichard, Wagner and coworkers describes the development and evaluation of antibacterial peptides that target the sliding clamp (SC) and the ribosome. The study is thoroughly performed and the manuscript is very well laid out and clearly communicated.
With the growing importance for developing alternative strategies to target resistant bacteria, i highly recommend acceptance of this excellent study after minor revision noted below.

Minor points:
1. Table 2 appears to have its caption/footnotes missing.

Answer:
We have corrected this point (see above, answer to the EDITORS COMMENT)

2. Neither in Fig 3 nor by the full images shown in the Supplementary Information could I find mentioned how many times this experiment were repeated. At least two biological replicates should be performed.

Answer:
We modified the legend to Figure 3 in the main text (Page 9, left column) to make it clear that the FITC-Py-P7 mediated co-immunoprecipitation of the SC was confirmed in three independent bilogical replicates.
"Full gel image and total protein stain of this representative assay are shown in SI.5. The specific FITC-Py-P7 mediated co-immunoprecipitation of the SC was confirmed in 3 independent biological replicates."

3. HR-MS analyses should be provided for the synthesised peptides.

Answer:
We have performed the HRMS analyses of all the fusion peptides synthezised in this study and provide the High Resolution mass spectrometry analyses spectra within the ESI supplementary information file (SI. 1b). We have modified the text accordingly (Page 1: "Electronic Supplementary Information (ESI) available: [SI.1a: HPLC and LCMS analyses of the different peptides synthesized. SI.1b: HRMS analyses of the different peptides synthesized. SI.3: Typical ITC titration curves describing the interaction of the different peptides with EcSC and Ec70S. SI.4: Analysis of the interaction of the different peptides with EcSC and Ec70S. SI.5: Whole gel images of the co-immunoprecipitation assays. SI.6: Survival curves of E. coli infected D. melanogaster from three independent experiments.]"; Page 3: "HPLC and LCMS analyses of all peptides synthesized are presented in SI.1a. HRMS analyses of all the fusion peptides synthesized are presented in SI.1b."


Other modifications:
Some typographic errors were corrected within the author contribution paragraph, page 10:
"D. B., G. G., J-M. R., F.V. and J.E. W. analyzed the data. D. B., G. G. and J.E. W. wrote the paper."

Berlin, July 6, 2020

Dear Dr Wagner:

Manuscript ID: CB-ART-05-2020-000060.R1
TITLE: Antibacterial Activity of a Dual Peptide Targeting the Escherichia coli Sliding Clamp and the Ribosome

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

Reviewer 2

I have no further comments and recommend acceptance of the author's revised version.