Examination of yield, bacteriolytic activity and cold storage of linker deletion mutants based on endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6

Sosuke Munetomo; Jumpei Uchiyama; Iyo Takemura-Uchiyama; Thamonwan Wanganuttara; Yumiko Yamamoto; Toshihiro Tsukui; Hideharu Hagiya; Shuji Kanamaru; Hideyuki Kanda; Osamu Matsushita

doi:10.1371/journal.pone.0310962

Peer Review History

Original SubmissionApril 28, 2024
23 Jul 2024 Decision Letter - Kornelius Zeth, Editor PONE-D-24-17026Examination of yield, bacteriolytic activity and cold storage of linker deletion mutants based on endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6PLOS ONE Dear Dr. Uchiyama, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ==============================Both reviewers consider your manuscript as a valuable contribution to the field of endolysin research. However, there are minor changes to be done for the authors before the manuscript can be published. 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Munetomo et al., characterized three linker deletion mutants ORF93-delta05, ORF93-delta10, and ORF93-delta15 of endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6. I agree with Authors that optimization of the linker sequence is necessary to obtain a desirable chimeric protein. There are a lot of studies showing that the optimal linker sequence length is crucial for high protein activity (see for example https://doi.org/10.1007/s00253-008-1468-4, https://doi.org/10.1016/j.molimm.2003.08.006, https://doi.org/10.3390/pr8121587 or other). Therefore, I support publication of the manuscript in the PLOS ONE Journal after minor revision. Specific comments: ‘Amino acid residues’ sounds strange, 'amino acids' or 'residues', please correct throughout the manuscript, line 95, 99, 100 etc. The authors state that ‘the molecular masses of ORF93, ORF93-delta05, ORF93-delta10, and ORF93-delta15 were estimated to be 28.6 kDa, 27.9 kDa, 27.4 kDa, and 26.9 kDa, respectively’ or ‘the theoretical molecular masses were 31.3 kDa, 30.7 kDa, 30.2 kDa, and 29.7 kDa’. What programs were used to determine the molecular masses of studied proteins? What was the concentration of proteins used in bacteriolytic activity tests?, line 184 Reviewer #2: In the context of the prevalence of antibiotic resistance, it is crucial to develop alternative antibacterial strategies, and phage lysin is a good candidate molecule. However, there are many limiting factors in the development of phage lysins at present, and more basic research is needed. This work examined the influence of linker modification on the characteristics of the endolysisn derived from Staphylococcus jumbo phage S6, and found that ORF93-△15 showed the highest yield and bacteriolytic activity with a slight effect on the cold storage performance. Several concerns: 1. Figure 1 and Figure 2 need to be merged into one image because they illustrate the same problem, so there is no need to separate them into two separate images. 2. How long can the modified protein maintain activity at room temperature (e.g. 23 degrees)? 3. It is recommended to conduct further experiments on the modified content elements, such as temperature stability, pH stability tests, biofilm removal tests, in vivo antibacterial tests, etc. 4. In addition, how does extending the linker, altering or optimizing the linker's amino acid sequence affect lysin? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Shuguang Lu ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0310962.r001
Revision 1
2 Sep 2024 Author Response Responses to Reviewer #1’s comments We have responded to one general comment and three specific ones and revised the manuscript accordingly. Response to the general comment 1. Reviewer #1 recommended including some references supporting the fact that the optimal linker is crucial to the protein function. We have revised the manuscript to strengthen our manuscript as follows, with the addition of the suggested references. Line 82: “The influence of linker … been examined well, to our knowledge.” has been replaced with the following sentences. “For example, the expression of protein without a linker was not successful in Escherichia coli [20]. Changing the codons of the linker improved the expression of fusion protein [21]. The linker length is shown to be an important factor for protein characteristics such as stability [22], activity [23], and catalytic efficiency [24]. However, the influence of linker length on the endolysin characteristics has not been examined well, to our knowledge.” Line 88: “Escherichia coli” has been replaced with “E. coli.” Response to specific comments 2. Reviewer #1 suggested that “amino acid residues” be replaced with “amino acids.” We agree with this suggestion and have made this change throughout the text. Lines 94, 95, 99-100, 106, 111, 115, 122, and 138: “amino acid residue” has been replaced with “amino acid”. Lines 35, 100-101, 109, 111, 122, and 264: “amino acid residues” has been replaced with “amino acids”. 3. Reviewer #1 requested us to mention the software used to determine the molecular masses of studied proteins. We have modified the manuscript as follows. Line 383: The following sentence has been added. “The theoretical molecular mass was calculated from the protein sequence using GENETYX® Ver. 14.1.1 (Nihon server, Tokyo, Japan); the protein molecular mass was estimated from the thickest band in the SDS-PAGE image using ImageJ software (version 1.54g) [58].” 4. Reviewer #1 requested us to mention the protein quantity used in bacteriolytic activity tests in line 184. We have modified the manuscript as follows. Line 184: “In the assay, the endolysin … examined after incubation.” has been replaced with the following sentences. “In the assay, the endolysin solutions (3 µL of 0.1 mg/mL) were applied on the surface of double-layered culture media containing bacteria, and the transparency of the spot formed on the agar plate was examined after incubation.” Responses to Reviewer #2’s comments Reviewer #2 made 4 comments. We have responded to these comments and revised the manuscript accordingly. 1. Reviewer #2 suggested merging Figs 1 and 2. We have done so, and this is now included as a new Fig 1. We have modified the manuscript as follows. Line 114: “Fig 1. Description of ORF93. (A) Schematic … correspond to Fig 1A.” has been replaced with the following sentences. “Fig 1. Description of ORF93 and its mutants. The domains and linker are colored as follows: CHAP domain in green, linker in red, and SH3 domain in yellow. (A) Schematic diagram of ORF93 based on the primary structure. Each number represents the amino acid position from the N-terminus at both ends of the domain, counted from the N-terminus. (B) Predicted protein structure of ORF93. (C) Schematic diagram of ORF93 mutants based on the primary structure. The linker deletions are shown as shadowed areas. The numbers around shadowed areas represent the amino acid position from the N-terminus at both ends of the deletion area. ORF93-Δ05, ORF93-Δ10, ORF93-Δ15, and ORF93-Δ20 are the ORF93 mutants which 5, 10, 15, and 20 amino acids were deleted in the upstream direction from the 157th amino acid from the N-terminus. The predicted linker lengths are 21, 16, 11, and 6 amino acids, respectively. (D) Predicted protein structures of ORF93-Δ05, ORF93-Δ10 and ORF93-Δ15. These protein structures were overlapped based on the CHAP domain. (E) Predicted protein structure of ORF93-Δ20.” Line 121: “Fig 2A” has been replaced with “Fig 1C”. Line 127: “Fig 2B” has been replaced with “Fig 1D”. Line 129: “Fig 2C” has been replaced with “Fig 1E”. Line 132: “Fig 2. Description of ORF93 mutants. The domains and linker … protein structure of ORF93-Δ20.” has been deleted. Line 151: “Fig 3A” has been replaced with “Fig 2A”. Line 160: “Fig 3” has been replaced with “Fig 2”. Line 175: “Fig 3B” has been replaced with “Fig 2B”. Line 214: “Fig 4” has been replaced with “Fig 3”. Line 222: “Figure 4” has been replaced with “Fig 3”. Line 232: “Fig 5A” has been replaced with “Fig 4A”. Line 236: “Fig 5B” has been replaced with “Fig 4B”. Line 241: “Fig 5C” has been replaced with “Fig 4C”. Line 243: “Fig 5” has been replaced with “Fig 4” 2. Reviewer #2 addressed the stability of the proteins at room temperature (i.e., how long can the modified protein maintain activity at room temperature?). We appreciate this comment. The storability can extend the usability of endolysins. We consider this information to be highly appropriate when discussing general endolysin characteristics. Thus, we have modified the manuscript as follows. Line 322: A new paragraph has been added as follows. “To study the feasibility of endolysins, they must be investigated from other aspects, such as physical characteristics and therapeutic effects in vivo. First, physical characteristics such as pH stability and thermostability are important to develop an endolysin drug, because protein instability leads to irreversible aggregation and is a major concern in the development of new biopharmaceuticals. In particular, these characteristics have been investigated in detail to develop monoclonal antibodies as biopharmaceuticals [52, 53]. Similarly to this, the physical characteristics of endolysins must be investigated regarding their suitability in biopharmaceuticals in the future. In addition, the additives enabling the protein stability are also important, to extend the usability to an endolysin drug. For example, a previous study has shown that the activity of LysK endolysin was maintained at 100% when stored at 22 °C for one month with polyols such as glycerol, whereas without polyols, it decreased to 5% [54]. Moreover, the therapeutic effects in vivo may be varied depending on endolysin type. In the previous study, some endolysins have shown therapeutic effects equivalent to vancomycin in mice with systemic MRSA infections, whereas other endolysins have not [12]. In addition, it has been shown that endolysins exhibit different levels of biofilm removal activity [12]. We need to investigate the therapeutic effects in vivo with an understanding of the biochemical properties of endolysins.” 3. Reviewer #2 recommended conducting further experiments, such as temperature stability, pH stability tests, biofilm removal tests, in vivo antibacterial tests, etc. We appreciate these comments. However, our intention is to conduct these experiments in the future, because of the limits to the manuscript length and thus its content. Instead, we have reviewed the work on endolysins again, and have added these points to the new paragraph described right above in the response to comment No. 2 of Reviewer #2. 4. Reviewer #2 raised questions on the effects of extending the linker, and of altering or optimizing the linker’s amino acid sequence on endolysin function. We have modified the manuscript as follows. Lines 317–321: “Moreover, phage S6 … to maximize endolysin function [14-16, 32, 33, 40, 47]” has been replaced with “In addition, phage S6 is a giant phage, and a longer linker of ORF93 may produce larger holes in the peptidoglycan by simply extending the distance from CHAP to the SH3 domain.” Line 321: A new paragraph has been added as follows. “To understand the role of the linker further, it is also important to examine both linker extension and modification. A previous study showed that a longer linker improves the thermostability of subunit-fused nitrile hydratase; however, an excessively long linker can decrease its activity and expression [47], which is consistent with the results of this study. Moreover, in protein engineering, various linkers have been designed and classified as flexible, rigid, helical, and cleavable linkers [19, 49-51]. It has been reported that changing the type of linker can change the stability and activity of the enzyme [38, 47, 48]. In the future, the linker types will be examined in ORF93-Δ15.” Minor modifications Through this revision, we have found minor mistakes in the abstract. Thus, we have modified our manuscript as follows. Line 32: "linker modification" has been replaced with "linker shortening". Line 36: "all the endolysins" has been replaced with "ORF93 and its mutants". Lines 658–659: “The vertical and horizontal … of ORF93, respectively” has been replaced with “In the PAE, the scored residue x with regards to the aligned residue y is shown as a heatmap”. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0310962.r002
11 Sep 2024 Decision Letter - Kornelius Zeth, Editor Examination of yield, bacteriolytic activity and cold storage of linker deletion mutants based on endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6 PONE-D-24-17026R1 Dear Dr. Uchiyama, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Kornelius Zeth, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0310962.r003
Formally Accepted
11 Oct 2024 Acceptance Letter - Kornelius Zeth, Editor PONE-D-24-17026R1 PLOS ONE Dear Dr. Uchiyama, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Kornelius Zeth Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0310962.r004

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