Physicochemical and Proteolytic Barriers Limiting Activity of Cpl-1 and Pal Endolysins in Human Circulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The abstract states “The main physicochemical and biochemical factors limiting the activity and stability of cpl-1 and Pal endolysins in blood”. This paper clearly shows work to explore factors affecting the endolysins in blood, leading to the design of a mutant with better performance. 

 

Intro 

Appropriate background to AMR and introduction to the lysins role as a potential treatment. Good introduction to what is covered in the paper and justifications for their focus.

Challenge in avoiding the proteases - what are the other pharmacokinetic pathways for elimination of the endolysins? Such as protein modification? Can the authors comment on the other pathways that protein therapies are eliminated via. 

Good explanation of assay used

No mention of other literature examples with similar work on lysins? Or other protein therapies? Can the authors comment on the novelty of their work? And provide reference to any related studies in improving lysin stability or other protein stability in blood. 

 

Methods

Thorough description of the experimental protocols. All relevant information including suppliers is included.

 

Results

Why is the lytic activity enhanced in low plasma concentrations? Can the authors comment on this (in the discussion) as to any reasons why this occurs?

What in PBS is hindering Pal activity? (50% PBS result fig 3) The authors mention ionic strength, is it simply that or is it specific to the components of PBS?

Can the authors comment on whether there are any known interactions of Cpl or Pal with BSA? Did they carry out any binding assays to explore this? 

Why was PBS used? Would a carbonate buffer not be more relevant for blood comparisons?

Good description of in silico work.

Author Response

Comment 1

Appropriate background to AMR and introduction to the lysins role as a potential treatment. Good introduction to what is covered in the paper and justifications for their focus.

Challenge in avoiding the proteases - what are the other pharmacokinetic pathways for elimination of the endolysins? Such as protein modification? Can the authors comment on the other pathways that protein therapies are eliminated via. 

Response 1: We fully agree with the Reviewer that expanding on these aspects would strengthen the manuscript. We propose extended discussion of this problem in the Discussion section, aligning it with other complementary aspects (lines 598-609).

Comment 2

No mention of other literature examples with similar work on lysins? Or other protein therapies? Can the authors comment on the novelty of their work? And provide reference to any related studies in improving lysin stability or other protein stability in blood. 

Response 2: Thank you for this suggestion, we fully agree with the Reviewer. We have now added a fragment addressing these aspects (lines 39-54).

 

Comment 3

Why is the lytic activity enhanced in low plasma concentrations? Can the authors comment on this (in the discussion) as to any reasons why this occurs?

Response 3: We confirm that the enhancement of activity at low plasma concentrations was not expected and it represents a novel observation. We noted that the enhancement of endolysin activity in diluted serum is, on average, consistent with the effects exerted by bystander proteins, such as albumin (Fig. 5), which also enhanced activity at concentrations lower than those present in blood. At the same time, the endolysins investigated proved to be insensitive to dilutions of ions (Fig. 4). Therefore, we believe that diluted serum provides optimal conditions for bacteriolytic activity of endolysins. This finding underscores the heterogenous nature of plasma, where synergistic and antagonistic effects occur simultaneously and dynamically counterbalance each other. We have extended discussion to address this problem (lines 528-538).

Comment 4

What in PBS is hindering Pal activity? (50% PBS result fig 3) The authors mention ionic strength, is it simply that or is it specific to the components of PBS?

Response 4: We are grateful for drawing our attention to this interesting problem. We believe that that lower ion concentration without changing relative ratios of ions is affecting electrostatic interactions between protein, most probably cell-wall binding domain and bacterial cell surface. The effect is generally referred to as a shielding effect. This is probably why PBS seems to hinder Pal activity when compared to diluted PBS. We expanded our description of this issue in lines 80-98.

Comment 5

Can the authors comment on whether there are any known interactions of Cpl or Pal with BSA? Did they carry out any binding assays to explore this?

Response 5: We are grateful for this comment and we propose an improved discussion of this aspect in the manuscript (lines: 565-580).

Reviewer 2 Report

Comments and Suggestions for Authors

The study provides a high-quality, systematic evaluation of how the physiological environment (ions, proteins, and proteases) inhibits endolysin activity. The transition from identifying these barriers to engineering a protease-resistant variant (Pal16) is scientifically sound and therapeutically relevant.

However, the manuscript currently lacks fundamental enzymatic characterization. While the authors' "real-time lysis assay" is excellent for demonstrating functional success in blood-like conditions, it does not isolate the intrinsic catalytic efficiency of the engineered variants from their environmental resistance but you must address the following points:

1. Provide standard biochemical kinetic parameters (k_cat, K_m and k_cat/K_m) for the wild-type enzymes and the lead variants (such as Pal16). This should be performed using a purified substrate (such as purified peptidoglycan or a synthetic muropeptide) in a standardized buffer.

2. Use this data to clarify whether the mutations improved the enzyme's "engine" (catalytic power) or simply its "armor" (stability/resistance to inhibition). 

3. Update the Discussion section to reflect how these kinetic parameters correlate with the results observed in the Sytox Green lytic assays and the ex vivo mouse model.

4. The manuscript contains numerous grammatical errors must be revised and improved

 

Author Response

The study provides a high-quality, systematic evaluation of how the physiological environment (ions, proteins, and proteases) inhibits endolysin activity. The transition from identifying these barriers to engineering a protease-resistant variant (Pal16) is scientifically sound and therapeutically relevant.

Comments 1-3

However, the manuscript currently lacks fundamental enzymatic characterization. While the authors' "real-time lysis assay" is excellent for demonstrating functional success in blood-like conditions, it does not isolate the intrinsic catalytic efficiency of the engineered variants from their environmental resistance but you must address the following points:

1. Provide standard biochemical kinetic parameters (k_cat, K_mand k_cat/K_m) for the wild-type enzymes and the lead variants (such as Pal16). This should be performed using a purified substrate (such as purified peptidoglycan or a synthetic muropeptide) in a standardized buffer.
2. Use this data to clarify whether the mutations improved the enzyme's "engine" (catalytic power) or simply its "armor" (stability/resistance to inhibition).
3. Update the Discussion section to reflect how these kinetic parameters correlate with the results observed in the Sytox Green lytic assays and the ex vivomouse model.

Response to comments 1-3: We are grateful for this insightful comment and valuable advice, and we fully agree with the Reviewer. Unfortunately, at this stage we are unable to extend the experimental data as suggested due to significant limitations, including the completion of project funding and the fact that the principal investigator and project originator had to transition to another institution, which significantly limits our possibilities of additional experimental work. We would like, however, to underline that we highly appreciate the scientific merit of the suggestion and the perspective it brings. In the current manuscript, we have therefore chosen to address the issue thoroughly in the Discussion section, highlighting the limitations and context raised by the Reviewer, so that readers can fully understand the implications. We hope to pursue the recommended experiments in future work. (lines 610-658)

Comment 4:

The manuscript contains numerous grammatical errors must be revised and improved

Response 4: We apologize for the errors identified in the manuscript. We have made every effort to correct them and hope that the revised version is now substantially improved. Multiple corrections were made throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

 I recommend the article for publishing in present form.

Author Response

The authors are extremely grateful to the Reviewer for his work and (previous) remarks, which significantly improved the article.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Now, paper present in acceptable form