Efficient screening of adsorbed receptors for Salmonella phage LP31 and identification of receptor-binding protein

ABSTRACT The adsorption process is the first step in the lifecycle of phages and plays a decisive role in the entire infection process. Identifying the adsorption mechanism of phages not only makes phage therapy more precise and efficient but also enables the exploration of other potential applications and modifications of phages. Phage LP31 can lyse multiple Salmonella serotypes, efficiently clearing biofilms formed by Salmonella enterica serovar Enteritidis (S. Enteritidis) and significantly reducing the concentration of S. Enteritidis in chicken feces. Therefore, LP31 has great potential for many practical applications. In this study, we established an efficient screening method for phage infection-related genes and identified a total of 10 genes related to the adsorption process of phage LP31. After the construction of strain C50041ΔrfaL 58−358, it was found that the knockout strain had a rough phenotype as an O-antigen-deficient strain. Adsorption rate and transmission electron microscopy experiments showed that the receptor for phage LP31 was the O9 antigen of S. Enteritidis. Homology comparison and adsorption experiments confirmed that the tail fiber protein Lp35 of phage LP31 participated in the adsorption process as a receptor-binding protein. IMPORTANCE A full understanding of the interaction between phages and their receptors can help with the development of phage-related products. Phages like LP31 with the tail fiber protein Lp35, or a closely related protein, have been reported to effectively recognize and infect multiple Salmonella serotypes. However, the role of these proteins in phage infection has not been previously described. In this study, we established an efficient screening method to detect phage adsorption to host receptors. We found that phage LP31 can utilize its tail fiber protein Lp35 to adsorb to the O9 antigen of S. Enteritidis, initiating the infection process. This study provides a great model system for further studies of how a phage-encoded receptor-binding protein (RBP) interacts with its host's RBP binding target, and this new model offers opportunities for further theoretical and experimental studies to understand the infection mechanism of phages.


Sincerely, Olaya Rendueles
Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): Ge and colleagues present a novel method to elucidate the receptor binding site of phage LP31, infectious for specific strains of Salmonella enterica.Ge et al. constructed a random insertion transposon mutant library to identify phage resistant LPS mutants, and then created knockout strains which were subjected to agglutination, phage adsorption studies, and TEM confirm that the O9 antigen was the bacterial phage receptor site.Gu et al. also carried out further adsorption studies to identify phage protein Lp35, a tail fibre protein, involved in the binding of phage LP31 to O9.This work is timely given the rise in drug-resistant Salmonella spp. in many locations worldwide.Moreover, the techniques presented could be applied to many different phagebacteria combinations to accelerate phage therapy.The manuscript is well written, the methods well described, and the work carried out to a high standard.Therefore, I only have minor comments (detailed below).This manuscript describes a nice and thorough approach for identifying the receptor for phage, demonstrating that the Salmonella phage LP31 adsorbs to the O9 antigen in the outer membrane.A few minor comments: It would be useful to provide references for phage resistance caused by blocking injection (line 79) because this process is less commonly recognized vs other mechanisms (Salmonella phage P22 is a good example).The source of phage would be useful, (including vB_ SenS_SE1) for others trying to replicate this work.On line 199 a brief sullary of how you "optimized " yhe process would be helpful for readers.On line 201, "resistant" would be more appropriate than resistance.On line 255 ALl mutants are genetic, substitute "deletion mutant"

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin the revision process.The information that you entered when you first submitted the paper will be displayed.Please update the information as necessary.Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Microbiology Spectrum
I wish to submit our revised manuscript entitled "Efficient screening of adsorbed receptor for Salmonella phage LP31 and identification of receptor binding protein" for publication in Microbiology Spectrum.
We are grateful to the reviewers for such a rapid response to our submission, as well as their helpful comments and suggestions, which have helped us improve our manuscript.We have revised the manuscript based on all of the comments and have provided our point-by-point responses to each of their comments below.All changes are marked in yellow.
We hope that the revised manuscript is now suitable for publication in your journal.
Thank you and I look forward to hearing from you.

Sincerely
Xiang Chen, E-mail: chenxiang@yzu.edu.cnReviewer #1: Ge and colleagues present a novel method to elucidate the receptor binding site of phage LP31, infectious for specific strains of Salmonella enterica.Ge et al. constructed a random insertion transposon mutant library to identify phage resistant LPS mutants, and then created knockout strains which were subjected to agglutination, phage adsorption studies, and TEM confirm that the O 9 antigen was the bacterial phage receptor site.Ge et al. also carried out further adsorption studies to identify phage protein Lp35, a tail fibre protein, involved in the binding of phage LP31 to O 9 .This work is timely given the rise in drug-resistant Salmonella spp. in many locations worldwide.Moreover, the techniques presented could be applied to many different phage-bacteria combinations to accelerate phage therapy.The manuscript is well written, the methods well described, and the work carried out to a high standard.Therefore, I only have minor comments (detailed below).Line 367: Do the 'replicates' here refer to bootstraps?Please clarify this.

Reviewer #2:
This manuscript describes a nice and thorough approach for identifying the receptor for phage, demonstrating that the Salmonella phage LP31 adsorbs to the O9 antigen in the outer membrane.A few minor comments: It would be useful to provide references for phage resistance caused by blocking injection (line 79) because this process is less commonly recognized vs other mechanisms (Salmonella phage P22 is a good example).
Thank you so much for your suggestion.I have supplemented the references on blocking genomic injection according to your suggestion (line 79).
The source of phage would be useful, (including vB_ SenS_SE1) for others trying to replicate this work.
In Fig. 5A, I have indicated the Genebank numbers of all phages used for sequence analysis (line 591).Additionally, I have added references to the phage vB_SenS_SE1 according to your suggestion (line 171).
On line 199 a brief sullary of how you "optimized " the process would be helpful for readers.
Thank you for your suggestion.I have created Fig. 6 and provided a detailed description of the screening process.In addition, I have supplemented the optimization step (line 195) as suggested.
On line 201, "resistant" would be more appropriate than resistance.
Thank you for your suggestion.I have replaced "resistance" with "resistant" (line 197).
On line 255 All mutants are genetic, substitute "deletion mutant" Thank you for your suggestion.I have replaced " genetic mutant " with " deletion mutant " (line 251).Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
The ASM Journals program strives for constant improvement in our submission and publication process.Please tell us how we can improve your experience by taking this quick Author Survey.

Publication Fees:
We have partnered with Copyright Clearance Center to collect author charges.You will soon receive a message from no-reply@copyright.com with further instructions.For questions related to paying charges through RightsLink, please contact Copyright Clearance Center by email at ASM_Support@copyright.com or toll free at +1.877.622.5543.Hours of operation: 24 hours per day, 7 days per week.Copyright Clearance Center makes every attempt to respond to all emails within 24 hours.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Spectrum.Sincerely, Olaya Rendueles Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org

Fig 2 :
Fig 2: The yellow tones are difficult to read.Please consider replacing these with colours that provide more contrast.Fig 4: Please consider adding descriptive text to clarify the meaning of the arrows (i.e. that they are pointing at the phages).Please also consider indicating what protein Lp35 is as the figure appears before the text describing this in the manuscript.Line 367: Do the 'replicates' here refer to bootstraps?Please clarify this.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

Fig 2 :
Fig 2: The yellow tones are difficult to read.Please consider replacing these with colours that provide more contrast.Thank you very much for your suggestion.I have replaced the yellow in Fig. 2 with golden brown colour.

Fig 4 :
Fig 4: Please consider adding descriptive text to clarify the meaning of the arrows (i.e. that they are pointing at the phages).Please also consider indicating what protein Lp35 is as the figure appears before the text describing this in the manuscript.Thank you for your suggestion.I have added in the article that the arrow in the figure points to the phage (line 574).In addition, I have also marked the LP35 protein as the tail protein (RBP) of phage LP31 (lines 577-578).
-23R1 (Efficient screening of adsorbed receptor for Salmonella phage LP31 and identification of receptor binding protein) Dear Prof. Xiang Chen: