Proteomic Analysis of Thermus thermophilus Cells after Treatment with Antimicrobial Peptide

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The issues mentioned in the 1st round review were fixed and the paper now is relatively sound and ready to go. 

Author Response

Comments: The issues mentioned in the 1st round review were fixed and the paper now is relatively sound and ready to go. 

Answer: We are very grateful to the reviewer for the comments and suggestions.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

In this manuscript, the authors investigate the proteome alterations of Thermus thermophilus exposed to three concentrations of the artificial amyloidogenic antimicrobial peptide R23I. The goal of using both inhibitory and non-inhibitory concentrations was to differentiate between the stress response mechanisms and the peptide's inhibitory effects, which are presumed to target protein synthesis. They identified 82 proteins, including both up-regulated and down-regulated, encompassing various membrane proteins, ribosomal proteins, and Krebs cycle enzymes.

The topic is highly relevant due to the ongoing issue of antibiotic resistance. The objective is clear, and the experimental plan is both solid and well-structured. However, a key aspect is missing from the results: the comparison between inhibitory and non-inhibitory doses. From a certain point onward, specifically in figures 4 and 5, this comparison is not adequately addressed, leaving the interpretation of the results incomplete.

For example, are the ribosomal proteins, membrane proteins (as shown in Figure 5), and tricarboxylic cycle enzymes regulated differently in the 20 µg/ml dose group compared to the 50 and 100 µg/ml groups?

At inhibitory concentrations (50 and 100 µg/ml), shouldn't there be a generalized downregulation if the amyloidogenic peptide R23I is inhibiting protein synthesis?

The discussion is somewhat lacking and would benefit from more engagement with the existing literature. The methodological details should be streamlined and not presented as the concluding point of the discussion. Additionally, are there proteins identified in the previous work (reference 23, Kurpe SR et al. 2020) that were also identified using the new software, compared to the old one?

Regarding the treatment of bacteria with R23I, the incubation times are not specified, which is crucial for determining whether the peptide's effects are immediate and direct or indirect as a result of other events. Moreover, the response curve (Figure 2) differs significantly from that in the previous work (reference 23, Kurpe SR et al. 2020). What accounts for this discrepancy? Were the incubation times the same?

In Figure 3C, the Heatmap of log2 ratios among the various groups is presented. Is the comparison made relative to the control sample (untreated)? If so, why are there both positive and negative variations in the control column? What do these variations represent?

Minor Points:

• Lane 130: An introductory sentence on proteomic analysis would be helpful for the reader. The text transitions from discussing the treatment of bacteria with the peptide R23I to peptides as proteolytic fragments in proteomic analysis in the next sentence without clarification.

• Lane 224: I am unsure about the role of bacterial RNA polymerase in this context.

Comments on the Quality of English Language

I am not a native English speaker, but it seems to me that the English is good.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

I have read the authors' response letter, which I appreciate. Many points have been addressed, but some responses have raised new questions.

Answer: We are very grateful to the reviewer for the valuable comments and suggestions.

 

........... However, a key aspect is missing from the results: the comparison between inhibitory and non-inhibitory doses. From a certain point onward, specifically in figures 4 and 5, this comparison is not adequately addressed, leaving the interpretation of the results incomplete.

For example, are the ribosomal proteins, membrane proteins (as shown in Figure 5), and tricarboxylic cycle enzymes regulated differently in the 20 µg/ml dose group compared to the 50 and 100 µg/ml groups?

At inhibitory concentrations (50 and 100 µg/ml), shouldn't there be a generalized downregulation if the amyloidogenic peptide R23I is inhibiting protein synthesis?

Answer: Of course, you are right, in the case of inhibitory concentrations we can observe a general decrease in protein synthesis. However, at an early stage after adding AMP, there is a gradual penetration of AMP into the cells. Thus, for some period of time, the cells or part of the cells are able to respond to the presence of AMP in the cell. We record the average time-based general response from all cells. Therefore, it is possible to observe the regulatory activity of the cells at an early stage after adding inhibitory concentrations of AMP.

Reviewer answer:  I appreciate the author's response. However, there is no experimental evidence supporting the "gradual penetration of peptides into the cells." As mentioned in the revised version, bacterial cells were incubated with the R23I peptide for 24 hours before the proteomic analysis—a duration that far exceeds the normal bacterial life cycle. Over 24 hours, the peptides would have had ample time to exert their effects.

 

The principal component analysis (PCA) clearly shows that the group treated with 20 µg/ml forms a distinct cluster from the groups treated with 50 and 100 µg/ml. At this subinhibitory concentration, could this reflect a stress response of the bacteria to the peptide? Based on the results in Figure 3, this does not appear to be the case, as it includes a number of proteins with lower abundance compared to the 50 and 100 µg/ml groups.

 

Another important point is the incubation temperature, which was not mentioned in the analysis. T. thermophilus grows at 50–85°C. What temperature was used in the incubation experiments with R23I?

 

The discussion is somewhat lacking and would benefit from more engagement with the existing literature. The methodological details should be streamlined and not presented as the concluding point of the discussion. Additionally, are there proteins identified in the previous work (reference 23, Kurpe SR et al. 2020) that were also identified using the new software, compared to the old one?

Answer: We have added new discussion and new references. In addition, we have added a new Figure 7 to compare new and old results (Venn diagrams for proteins found by the PEAKS and IdentiPy programs).

Reviewer answer: I appreciate the authors' revisions to the discussion. However, I still find the placement of the methodological comparison in the final part of the discussion inappropriate, even though the topic is interesting. Additionally, this section is not mentioned in the abstract. Please consider moving it to the beginning of the discussion or another more suitable section

.

Regarding the treatment of bacteria with R23I, the incubation times are not specified, which is crucial for determining whether the peptide's effects are immediate and direct or indirect as a result of other events. Moreover, the response curve (Figure 2) differs significantly from that in the previous work (reference 23, Kurpe SR et al. 2020). What accounts for this discrepancy? Were the incubation times the same?

Answer: At present, we cannot say whether the effect of AMP on cells occurs immediately and directly or indirectly. However, according to the heat map (Figs. 3 and 5), the concentration of 67 cellular proteins decreases with increasing AMP concentration, while the concentration of 15 cellular proteins increases.

Reviewer answer: I disagree with the last observation: the log2 ratio results (Figures 3C and 4) show a general decrease in the abundance of 67 proteins compared to the untreated controls, but there is no clear direct relationship between AMP concentration and protein decrease. For example, the S2 protein decreases in the 20 and 50 µg/ml groups but increases again at 100 µg/ml. Similarly, L24, S15, and L27 decrease at 20 and 100 µg/ml but increase at 50 µg/ml. Additionally, 15 proteins show increased abundance  but not in all concentration groups.

In both experiments the incubation time was 24 hours. We have added this information to the methods description.

I appreciate the authors for providing this important information. However, this choice should be justified. Typically, incubation times of 15 minutes to 2 hours are used to study immediate or early responses to stimuli (e.g., stress, antibiotics, or nutrient shifts), as changes in protein expression can be observed within 30 minutes to 2 hours in rapidly responding systems. Longer times are generally used for slow-growing bacteria. Is this the case here?

The apparent difference arises from the logarithmic axis of the peptide concentration. The curve shown in green in Figure 2 does not differ significantly from the curve shown in black, but rather refines the data obtained.

I thank the authors for this clarification, and I suggest replacing "unpublished data" in the figure 2  with "this study."

 

In Figure 3C, the Heatmap of log2 ratios among the various groups is presented. Is the comparison made relative to the control sample (untreated)? If so, why are there both positive and negative variations in the control column? What do these variations represent?

Answer: The heat map shown in Figure 3C plots the log2 ratios of each group’s abundance relative to the average protein abundance across all groups. Specifically, the average protein abundance in a group is divided by the average protein abundance across all groups, and then the logarithm to base 2 is taken according to the Peaks Studio algorithm.

"I thank the authors for the precise explanation that has been added to the Materials and Methods section."

Comments on the Quality of English Language

I have no comments to make.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article description is relatively comprehensive. However, it is compromised by the lack of certain specific experimental procedure or design consideration, thus affecting the persuasiveness of the conclusions drawn. If the author were to provide more in-depth information on the following points, it would greatly support the paper, making it more thorough.

1)      Iine 144-145, “changes in the proteome were identified by calculating the protein abundance fold changes (FC) with respect to the control condition.”  As we know the abundance of protein from MS relies on sample injection/loading amount. How did the author control the loading amount between the control and samples? depends on gel band intensity? or total peptide amount (ug) to inject into LC-MS? Or cell numbers when harvesting the sample and control? The specific question is how to make sure the abundance change is not a result of loading amount variation between control and sample?

2)      Line 230-240, the author talked about using SDS-PAGE under reducing conditions. In the early background part, the author also mentioned that this peptide is possible to co-aggregate with S30. Did the author observe any aggregation from samples? If so, can the aggregate all be reduced under the current SDS condition? In other words, if the peptide and ribosome form aggregation which can not be separated by current gel condition, it could introduce bias into the results analysis. The author should provide a more detailed explanation or present gel images to prove the result.

3)      Line 250-251, the author briefly says “Enzymatic hydrolysis was carried out by incubating the sample with a trypsin solution….”, There should be a reduction and alkylation process before trypsin digestion, right? Because in the result analysis section, the author mentioned "carbamidomethylation”

4)      Line 195-208, the author used S-layer protein as an example, especially making the conclusion that “with an increase in the concentration of the R23I peptide, a decrease 202 in the relative concentration of the S-layer protein was observed”. The author should present real data to demonstrate this.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript concerns proteomics data from treatment of T. thermophiles with an AMP that was isolated, analyzed and described in a previous publication (ref. 53 from 2020) from the group. The AMP R23I causes growth inhibition and aggregation of ribosomal protein S1. The present study does not add much to the LC/MS proteomic analysis reported in the previous publication, the different cell lysis procedure and sample treatment in the present study did not really improve the poor coverage in the work reported previously (291 here, 201 in ref 53). The data from the previous study is not at all included in a discussion of the present work. This new study, however, do report the identity of the proteins identified as having a significantly changed abundance in the cultures treated with the AMP, some mentioned in the text and some only in file S1.

The present manus also contains other types of data analysis and presentation. The heatmap in Fig. 3C is odd, it includes the 2 control samples and the 3 different samples from the treatments, not the average from the treatments (and illustrates a high sample variability for the highest concentration).

Fig. 4 show GO representations. Fig. 4 A is nonsense: first of all normal bacteria do not contain intracellular organelles, and what is the meaning of membrane, plasma membrane and outer membrane??.

Materials and methods is missing all information about how the “unpublished data” in Fig. 2 were obtained, about how the cells were grown for the LC/MS analysis (including whether it is biological replicates), and about the programs used for obtaining the presentations shown in Fig. 3 (and maybe Fig 4). And the data underlying the new estimation of antimicrobial effect should be included (in a supplementary file).

There is something wrong in the text with references 42-51, the references are in the list but they do not fit with the text where they are inserted.

There is an abundance of not really relevant references in the introduction.

Figure 3 suffers from a very small font of axes labels.

There is any real discussion of the results, e.g. in relation to the effect of inactivation of S1 protein, and maybe referring to papers about E. coli S1 (M A Sørensen 1, J Fricke, S Pedersen. 1998. Ribosomal protein S1 is required for translation of most, if not all, natural mRNAs in Escherichia coli in vivo. JMB 280: 561-9, and F Delvillani, G Papiani, G Deho` and F Briani. 2011. S1 ribosomal protein and the interplay between translation and mRNA decay. NAR 39 7702-7715. doi:10.1093/nar/gkr417) some hypothesis about why the ribosomal proteins in general are down regulated, or whether this is just due to the growth inhibition.

 

Comments on the Quality of English Language

The quality of the language is uneven throughout the manuscript. Fine in abstract, first half of introduction and in materials and methods, but needs some revision in the rest of the manus.